Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis

The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using ³¹P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.     

A new species of the spore genus Costatoperforosporites from Early Cretaceous deposits in Portugal and its taxonomic and palaeoenvironmental significance

A new species of spore, Costatoperforosporites friisiae sp. nov., is described from the Early Cretaceous (late Aptian–early Albian) deposits of Catefica in the Lusitanian Basin, western Portugal. Although the morphology of the muri is clearly of the Cicatricosisporites type, the presence of micropores on these sculptural elements and within the intervening grooves is a particularly distinctive feature and more typical of spores that have been attributed previously to Costatoperforosporites. As a result, this genus is emended not only to accommodate Costatoperforosporites friisiae but also to differentiate it more clearly from other, similar, murornate genera. It is considered to represent the family Anemiaceae. So far, Costatoperforosporites friisiae has only been encountered in the Catefica palynoflora. The presence of many other pteridophyte spores, especially of schizaealean derivation, together with abundant cheirolepidiacean remains at this locality, strongly suggests a warm, moist climate and diverse source vegetation.     

Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy

Scanning laser confocal microscopy was utilized to visualize sites of hydrogen peroxide release from stimulated neutrophils and lysosomal acid phosphatase in these and other cells using cerium in the detection systems. Imaging of the cerium-containing reactions was achieved by employing the reflectance mode of this instrument. Localization of these products at the light microscope level was direct and did not require other reactions to generate a visible product. This new approach to cerium cytochemistry should prove useful for many applications.     

Palynostratigraphic,palynofacies, organic geochemical and palaeoenvironmental analysis of the Silurian Tanezzuft Formation in the Ghadames Basin of north-west Libya

Two palynofacies associations are documented from the Silurian Tanezzuft Formation in the Ghadames Basin. These are characteristic of the basal ‘Hot Shale’ and the overlying deposits, referred to here as the Cold Shale. The former reflects deposition in distal suboxic anoxic conditions and is dominated by highly oil-prone amorphous organic matter (AOM) typical of deposition in generally anoxic, restricted marine basins. Only a few acritarchs, prasinophyte algae and chitinozoans occur in association: virtually no spores or cryptospores were recorded. Thick-walled prasinophytes are most numerous in this part of the Tanezzuft Formation in both the Ghadames and Murzuq basins, suggesting enhanced surface water productivity. Deposition took place after the melting of the Late Ordovician ice sheets, which led to a major marine transgression. The palynofacies recorded from the overlying ‘Cold Shale’ deposits indicate deposition in distal shelf and basin conditions that were also relatively anoxic. They contain more palynomorphs, especially acritarchs, and generally less AOM. The phytoplankton assemblages are dominated by simple and thin-walled prasinophyte algae (leiospheres), suggesting dysoxic–anoxic conditions. Overall the middle and the upper parts of the Tanezzuft Formation are regarded as being deposited in distal dysoxic–anoxic shelf, distal dysoxic–oxic shelf and distal suboxic–anoxic basin respectively. Because chitinozoans are very rare, age determinations of the samples investigated are based mainly on acritarchs. The Hot Shale is dated as early-mid Rhuddanian (early Llandovery) whereas the rest of the formation is considered to late Rhuddanian–Telychian in age. As documented previously from other samples of the Tanezzuft Formation in both the Ghadames and Murzuq basins, the Hot Shale has a very high TOC content and excellent source potential for liquid hydrocarbons, whereas rest of the formation is less rich in organic matter with larger terrestrial and oxidized components and hence reduced potential for sourcing hydrocarbons (both oil and gas).     

Susceptibility of white-tailed deer (Odocoileus virginianus) to experimental infection with epizootic hemorrhagic disease virus serotype 7

During the fall of 2006, in Israel, epizootic hemorrhagic disease virus (EHDV) serotype 7 caused an intense and widespread epizootic in domestic cattle that resulted in significant economic losses for the dairy industry. The susceptibility of potential North American vector and ruminant hosts to infection with EHDV-7 is not known but is essential to understanding the potential for establishment of this exotic orbivirus in North America if it were introduced. Our primary objective was to determine whether white-tailed deer (WTD; Odocoileus virginianus) are susceptible to infection with EHDV-7. Six, 8-mo-old WTD were experimentally infected with EHDV-7, and all became infected and exhibited varying degrees of clinical disease. Clinical signs, clinicopathologic abnormalities, and postmortem findings were consistent with previous reports of orbiviral hemorrhagic disease (HD) in this species. Four of six animals died or were euthanized because of the severity of disease, one on postinoculation day (PID) 5 and the remaining WTD on PID 7. All deer had detectable viremia on PID 3, which peaked on PID 5 or 6 and persisted for as long as PID 46 in one animal. Deer surviving the acute phase of the disease seroconverted by PID 10. Based on the 67% mortality rate we observed, this strain of EHDV-7 is virulent in WTD, reaffirming their role as a sentinel species for the detection of endemic and nonendemic EHDV. Further, the observed disease was indistinguishable from previous reports of disease caused by North American EHDV and bluetongue virus serotypes, highlighting the importance of serotype-specific diagnostics during suspected HD outbreaks.     

Invasion by <Emphasis Type="Italic">Aegilops triuncialis</Emphasis> (Barb Goatgrass) Slows Carbon and Nutrient Cycling in a Serpentine Grassland

Invasive plant species alter plant community composition and ecosystem function. In the United States, California native grasslands have been displaced almost completely by invasive annual grasses, with serpentine grasslands being one of the few remaining refugia for California grasslands. This study examined how the invasive annual grass, Aegilops triuncialis, has altered decomposition processes in a serpentine annual grassland. Our objectives were to (1) assess howA. triuncialis alters primary productivity and litter tissue chemistry, (2) determine whether A. triuncialis litter is more recalcitrant to decomposition than native litter, and (3) evaluate whether differences in the soil microbial community in A. triuncialis-invaded and native-dominated areas result in different decomposition rates of invasive and/or native plant litter. In invaded plant patches, A. triuncialis was approximately 50% of the total plant cover, in contrast to native plant patches in which A. triuncialis was not detected and native plants comprised over 90% of the total plant cover. End-of-season aboveground biomass was 2-fold higher in A. triuncialis dominated plots compared to native plots; however, there was no significant difference in belowground biomass. Both above- and below-ground plant litter from A. triuncialis plots had significantly higher lignin:N and C:N ratios and lower total N, P, and K than litter from native plant plots. Aboveground litter from native plots decomposed more rapidly than litter from A. triuncialis plots, although there was no difference in decomposition of belowground tissues. Soil microbial community composition associated with different soil patch types had no effect on decomposition rates. These data suggest that plant invasion impacts decomposition and nutrient cycling through changes in plant community tissue chemistry and biomass production.     

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

Background  

Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors.     

Two three-dimensional cyano-bridged bimetallic cadmium(II) and copper(II)-copper(I) mixed-valence polymer compounds: Crystal structures and thermal stability analysis and magnetic properties

Two cyano-bridged cadmium(II) and copper(II)-copper(I) mixed-valence polymer compounds 1 and [Cu^II(en)₂]_0.5[CdCu^I(CN)₃] · Cl · H₂O 2 (en = 1,2-ethanediamine) through systematically varying the solution environment have been synthesized and structurally characterized by single crystal X-ray diffraction analysis. The cyano anions connected the copper and cadmium centres with bidentate or tridentate bridging modes resulting in two complicated 3D networks for 1 and 2. Compound 1 has a rare 5,6-connected network, while compound 2 can be reduced to a 6-connected α-Po net. The magnetic and thermal properties of 1 and 2 are also studied.