European phylogeography of the coastal plants Cakile maritima Scop. (Brassicaceae) and Eryngium maritimum L. (Apiaceae)

Linear dispersal systems, such as coastal habitats, are well suited for phylogeographic studies because of their low spatial complexity compared to three dimensional habitats. Widely distributed coastal plant species additionally show azonal and often essentially continuous distributions. These properties, firstly, make it easier to reconstruct historical distributions of coastal plants and, secondly, make it more likely that present distributions contain both Quaternary refugia and recently colonized areas. Taken together this makes it easier to formulate phylogeographic hypotheses. This work investigated the phylogeography of Cakile maritima and Eryngium maritimum, two species growing in sandy habitats along the north Atlantic Ocean and the Mediterranean Sea coasts on two different spatial scales using AFLP data. The genetic structure of these species was investigated by sampling single individuals along most of their distributions from Turkey to south Sweden. On a regional scale the population genetic structure of both species was also studied in detail in the Bosporus and Dardanelles straits, the Strait of Gibraltar and along a continuous stretch of dunes in western France. Additionally, populations of C. maritima were investigated in the Baltic Sea/Kattegat/North Sea area. Over the complete sampling range the species show both differences and similarities in their genetic structure. In the Mediterranean Sea, both species contain Aegean Sea/Black Sea and west Mediterranean clusters. Cakile maritima additionally shows a clustering of Ionian Sea/Adriatic Sea collections. Further, both species show a subdivision of Atlantic Ocean/North Sea/Baltic Sea material from Mediterranean. Within the Atlantic Ocean group, C. maritima from the Baltic Sea and the most northern Atlantic localities form an additional cluster while no such substructure was found in E. maritimum. In all three instances where population genetic investigations of both species were performed in the same area, the results showed almost complete congruency of spatial genetic patterns. In the Aegean/Black Sea/Marmara region a subdivision of populations into a Black Sea, a Sea of Marmara and an Aegean Sea group is shared by both species. In addition the Sea of Marmara populations are more close to the Aegean Sea populations than they are to the Black Sea populations in both cases. Populations from the Atlantic side of the Strait of Gibraltar are differentiated from those on the Mediterranean side in both species, a pattern that confirms the results of the wide scale study. Along the dunes of West France no clear genetic structure could be detected in any of the species. Additionally, the results from the Baltic Sea/North Sea populations of C. maritima did not reveal any geographical genetic pattern. It is postulated that the many congruencies between the species are mainly due to a predominantly sea water mediated seed dispersal in both species and their shared sandy habitat. The results are compared to hypothetical distributions for the last glacial maximum based on species specific temperature requirements. It is argued that in both species the geographical borders of the clusters in the Mediterranean area were not affected by quaternary temperature changes and that the Aegean/Black Sea/Marmara cluster, and possibly the Ionian Sea/Adriatic Sea cluster in C. maritima, is the result of sea currents that isolate these basins from the rest of the sampled areas. The genetic gap in the Strait of Gibraltar between Atlantic Ocean and Mediterranean Sea populations in both species is also explained in terms of sea currents. The existence of three subgroups corresponding to the Aegean Sea, Black Sea and Sea of Marmara basins is suggested to have arisen due to geographical isolation during periods of global sea regressions in the glacials. The population genetic evidence was inconclusive regarding the Baltic Sea cluster of C. Maritima from the wide scale study. The results of this study are very similar to those of an investigation of three other coastal plant species over a similar range. This suggests that the phylo-geographic patterns of widespread coastal plants may be more predictable than those of other terrestrial plants.

10 Å cryo-EM structure of Drosophila melanogaster hexamerin and Limulus polyphemus hemocyanin

In the present study, the quaternary structures of Drosophila melanogaster hexamerin LSP-2 and Limulus polyphemus hemocyanin, both proteins from the hemocyanin superfamily, were elucidated to a 10 Å resolution with the technique of cryo-EM 3D-reconstruction. Furthermore, molecular modelling and rigid-body fitting allowed a detailed insight into the cryo-EM structures at atomic level. The results are summarised as follows: Hexamerin 1. The cryo-EM structure of Drosophila melanogaster hexamerin LSP-2 is the first quaternary structure of a protein from the group of the insect storage proteins. 2. The hexamerin LSP-2 is a hexamer of six bean-shaped subunits that occupy the corners of a trigonal antiprism, yielding a D3 (32) point-group symmetry. 3. Molecular modelling and rigid-body fitting of the hexamerin LSP-2 sequence showed a significant correlation between amino acid inserts in the primary structure and additional masses of the cryo-EM structure that are not present in the published quaternary structures of chelicerate and crustacean hemocyanins. 4. The cryo-EM structure of Drosophila melanogaster hexamerin LSP-2 confirms that the arthropod hexameric structure is applicable to insect storage proteins. Hemocyanin 1. The cryo-EM structure of the 8×6mer Limulus polyphemus hemocyanin is the highest resolved quaternary structure of an oligo-hexameric arthropod hemocyanin so far. 2. The hemocyanin is build of 48 bean-shaped subunits which are arranged in eight hexamers, yielding an 8×6mer with a D2 (222) point-group symmetry. The 'basic building blocks' are four 2×6mers that form two 4×6mers in an anti-parallel manner, latter aggregate 'face-to-face' to the 8×6mer. 3. The morphology of the 8×6mer was gauged and described very precisely on the basis of the cryo-EM structure. 4. Based on earlier topology studies of the eight different subunit types of Limulus polyphemus hemocyanin, eleven types of interhexamer interfaces have been identified that in the native 8×6mer sum up to 46 inter-hexamer bridges - 24 within the four 2×6mers, 10 to establish the two 4×6mers, and 12 to assemble the two 4×6mers into an 8×6mer. 5. Molecular modelling and rigid-body fitting of Limulus polyphemus and orthologous Erypelma californicum sequences allowed to assign very few amino acids to each of these interfaces. These amino acids now serve as candidates for the chemical bonds between the eight hexamers. 6. Most of the inter-hexamer contacts are conspicuously histidine-rich and evince constellations of amino acids that could constitute the basis for the allosteric interactions between the hexamers. 7. The cryo-EM structure of Limulus polyphemus hemocyanin opens the door to a fundamental understanding of the function of this highly cooperative protein.

Purification and Preliminary Characterization of the TM0727 Protein from Thermotoga maritima

The TM0727 gene of Thermotoga maritima is responsible for encoding what has been reported to be a modulator of DNA gyrase (pmbA). Although the function of pmbA is still unknown, it is believedto be involved in cell division, carbon storage regulation, and the synthesis of the antibiotic peptide microcin B17. It is suggested that it serves together with tldD, a known zinc dependent protease, tomodulate DNA gyrase. TM0727 is believed to be a zinc dependent protease that binds zinc in the central active site of the molecule, located between two equivalent monomeric units. However, thecrystal structure determined by Wilson et al. (2005) did not contain zinc. It therefore remains to be seen if TM0727 requires zinc for activity, or regulation, and if the protein is indeed a protease. To begin studying this protein, the gene was expressed in BL21(DE3) pLysS cells and the induction time was optimized. Using affinity and ion exchange chromatography, the protein has been successfully purified. The purification procedure can be replicated to obtain sufficient protein for characterization. Purification results show that the protein loses stability after 24 hours and remains stable under an imidazole-free lysis workup. Preliminary characterization of TM0727 has focused on understanding the protein’s structuralproperties through tryptophan fluorescence anisotropy measurements. The four tryptophan residues located within the TM0727 dimer fluoresce at different maximum wavelengths and with differentintensities upon excitation with 295nm light. These emission properties are highly sensitive to the environment (solvent, surrounding residues) of each tryptophan residue. The low number oftryptophans allows for a specific monitoring of the protein’s structure as it denatures. As more denaturant is added to the protein, its tryptophan environments have clearly altered. This is indicative of unfolding and increased solvent exposure of the protein. This unfolding has been confirmed with the addition of a fluorescent quencher. Additionally, fluorescence anisotropy measurements have been carried out on the protein to gain a preliminary understanding of the rotational dynamics of the tryptophan residues. These experiments excite the tryptophan residues within the sample using a polarized light source. Polarized emission is then detected, the degree of which depends on the rotational dynamics and local environment of the tryptophan residues. The protein was denatured and the changes in emission were recorded to detect these structural changes. Results have shown a large change in quaternary structure, consistent with a dimer to monomer transition, occurs at 1.5M Guandidine HCl. There has also been an examination of the crystal structure for the location of a potential active site. The inner cavity of the protein was inspected visually to locate a potential location for a catalytic triad, specifically the amino acids found in the active sites of serine, cyteine, and aspartateproteases. It was found that a potential aspartic protease active site may be located between the Asparate286 and Aspartate287 residues. Further investigation is warranted to test this remotepossibility.

Spatial and temporal analysis of land cover and landscape structure change in Zagros Forests

The Zagros oak forests in Western Iran are critically important to the sustainability of the region. These forests have undergone dramatic declines in recent decades. We evaluated the utility of the non-parametric Random Forest classification algorithm for land cover classification of Zagros landscapes, and selected the best spatial and spectral predictive variables. The algorithm resulted in high overall classification accuracies (>85%) and also equivalent classification accuracies for the datasets from the three different sensors. We evaluated the associations between trends in forest area and structure with trends in socioeconomic and climatic conditions, to identify the most likely driving forces creating deforestation and landscape structure change. We used available socioeconomic (urban and rural population, and rural income), and climatic (mean annual rainfall and mean annual temperature) data for two provinces in northern Zagros. The most correlated driving force of forest area loss was urban population, and climatic variables to a lesser extent. Landscape structure changes were more closely associated with rural population. We examined the effects of scale changes on the results from spatial pattern analysis. We assessed the impacts of eight years of protection in a protected area in northern Zagros at two different scales (both grain and extent). The effects of protection on the amount and structure of forests was scale dependent. We evaluated the nature and magnitude of changes in forest area and structure over the entire Zagros region from 1972 to 2009. We divided the Zagros region in 167 Landscape Units and developed two measures— Deforestation Sensitivity (DS) and Connectivity Sensitivity (CS) — for each landscape unit as the percent of the time steps that forest area and ECA experienced a decrease of greater than 10% in either measure. A considerable loss in forest area and connectivity was detected, but no sudden (nonlinear) changes were detected at the spatial and temporal scale of the study. Connectivity loss occurred more rapidly than forest loss due to the loss of connecting patches. More connectivity was lost in southern Zagros due to climatic differences and different forms of traditional land use.

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.

Circum-Antarctic Coastal Environmental Shifts During the Late Quaternary Reflected by Emerged Marine Deposits

This review assesses the circumpolar occurrence of emerged marine macrofossils and sediments from Antarctic coastal areas in relation to Late Quaternary climate changes. Radiocarbon ages of the macrofossils, which are interpreted in view of the complexities of the Antarctic marine radiocarbon reservoir and resolution of this dating technique, show a bimodal distribution. The data indicate that marine species inhabited coastal environments from at least 35000 to 20000 yr sp, during Marine Isotope Stage 3 when extensive iceberg calving created a 'meltwater lid' over the Southern Ocean. The general absence of these marine species from 20000 to 8500 yr sp coincides with the subsequent advance of the Antarctic ice sheets during the Last Glacial Maximum. Synchronous re-appearance of the Antarctic marine fossils in emerged beaches around the continent, all of wh ich have Holocene marine-limit elevations an order of magnitude lower than those in the Arctic, reflect minimal isostatic rebound as relative sea-level rise decelerated. Antarctic coastal marine habitat changes around the continent also coincided with increasing sea-ice extent and outlet glacial advances during the mid-Holocene. in view of the diverse environmental changes that occurred around the Earth during this period, it is suggested that Antarctic coastal areas were responding to a mid-Holocene climatic shift associated with the hydrological cycle. This synthesis of Late Quaternary emerged marine deposits demonstrates the application of evaluating circum-Antarctic phenomena from the glacial-terrestrial-marine transition zone.

Quaternary carbonate record of ODP Hole 115-709A

The CaCO3 content in Quaternary deep-sea sediments from Pacific and Atlantic oceans have been suggested to respond differently to glacial/interglacial cycles; CaCO3 contents are highest during glacials in the Pacific but highest during interglacials in the Atlantic Ocean. It is not yet clear as to whether a Pacific or an Atlantic pattern of CaCO3 fluctuations dominates the Indian Ocean. We have analyzed the Ocean Drilling Program (ODP) Site 709A from the western equatorial Indian Ocean for the last 1370 ka to determine the relationships between percentages and fluxes of CaCO3 and Quaternary paleoclimatic changes. We also analyzed the coarse (>25 µm) and fine (<25 µm) fractions of CaCO3 in an attempt at estimating the influence of differences in productivity of foraminifera and calcareous nannofossils in shaping the CaCO3 record. Carbon isotopes and Ba/Al ratios were used as indices of productivity. Percentages and fluxes of CaCO3 in the total sediment and <25 µm fraction do not show any clear relationships to glacial/interglacial cycles derived from d18O of the planktonic foraminifera Globigerinoides ruber. This indicates that CaCO3 fluctuations at this site do not show either a Pacific or an Atlantic pattern of CaCO3 fluctuations. Fluxes of CaCO3 (0.38 to 2.46 g/cm**2/ ka) in total sediment and Ba/Al ratios (0.58 to 3.93 g/cm**2/ka) show six-fold variability through the last 1370 ka, which points out that productivity changes are significant at this site. Fluxes of the fine CaCO3 component demonstrate a 26-fold change (0.02 to 0.52 g/cm**2/ka), whereas the coarse CaCO3 component exhibit eight-fold change (0.13 to 1.07 g/cm**2/ka). This suggests that productivity variations of calcareous nannofossils are greater in comparison with the foraminifera. On the other hand, mean values of coarse CaCO3 fluxes are higher compared to those of fine CaCO3, which reveals that the foraminifera contribute more to the bulk CaCO3 flux than the calcareous nannofossils in the equatorial Indian Ocean.