Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy

Using a new scheme based on atomic force microscopy (AFM), we investigate mechanical properties of clathrin-coated vesicles (CCVs). CCVs are multicomponent protein and lipid complexes of approximately 100 nm diameter that are implicated in many essential cell-trafficking processes. Our AFM imaging resolves clathrin lattice polygons and provides height deformation in quantitative response to AFM-substrate compression force. We model CCVs as multilayered elastic spherical shells and, from AFM measurements, estimate their bending rigidity to be 285 +/- 30 k(B)T, i.e., approximately 20 times that of either the outer clathrin cage or inner vesicle membrane. Further analysis reveals a flexible coupling between the clathrin coat and the membrane, a structural property whose modulation may affect vesicle biogenesis and cellular function.     

Cryopreservable neutrophil surrogates: granule-poor, motile cytoplasts from polymorphonuclear leukocytes home to inflammatory lesions in vivo

Cytokineplasts (CKP) are anucleate, motile, granule-poor fragments induced from polymorphonuclear leukocytes on surfaces by the brief application of heat. Derived from the peripheral cytoplasm and membranes of PMN, they retain the sensing, transducing, and effector mechanisms necessary for chemotaxis and phagocytosis, and appear to represent a functional, self-purification of the motile apparatus. Unlike their parent PMN, CKP are cryopreservable. We have shown that they can adhere to endothelial cell monolayers, open interendothelial cell junctions, and migrate to the abluminal side when stimulated by a chemoattractant. Employing an animal model, we now show that, given intravenously, they can home to an inflammatory target lesion in vivo.     

Detection and quantification of Erysiphe necator DNA in wine grapes and resultant must and juice

Powdery mildew of grapevines is difficult to assess visually at the weighbridge, particularly in large consignments of machine-harvested fruit. To facilitate accurate methods for the detection and quantification of the disease in grape samples obtained from both the vineyard and winery, we developed a DNA probe for the pathogen Erysiphe necator. The E. necator-specific 450 bp DNA fragment pEnA1, targets highly repetitive sequences and was isolated from a partial genomic library. In screening for species specificity, clone pEnA1 was used in slot-blot hybridization and detected E. necator DNA from grapes and resultant must and juice, but not from clarified juice and wine. The detection threshold was approximately 50 pg ofE. necator DNA per 100 ng total DNA of grape sample and was equivalent to 1–5 % of a grape bunch visually affected by powdery mildew. Disease severity, expressed as the percentage of surface area of a bunch with powdery mildew, and E. necator DNA content were highly correlated, r² = 0.955, P < 0.001. The DNA-based hybridization assay has the potential to predict the severity of powdery mildew in grape samples from the vineyard and in must and juice samples at the winery. The DNA sequence of clone pEnA1 was used to design species-specific primers, the results maintaining the same specificity patterns observed in the initial hybridization assays. The PCR-based assay was sensitive enough to detect approximately 1 pg DNA, being equivalent to 1 conidium per sample. This is the first report to date of the detection of all known phenetic groups of E. necator DNA and of the quantification of DNA from grape samples at the winery. Accurate information on the extent of powdery mildew contamination of grape lots would enable wineries to make more informed decisions about the use of fruit and must.     

Biodiversity and land use change on the Causse Méjan, France

This paper argues that due to the co-evolution of biological and cultural diversity, a meaningful study of biodiversity must be positioned within complex social-ecological systems. A complex systems framework is proposed for conceptualising the study of social-ecological systems. A case study approach is adopted whereby changes in biodiversity on the Causse Méjan, France, are linked with changes in society, land use, agricultural practices and policies. We argue that ecological and social resilience is linked through the dependence on ecosystems of communities, and in turn by the influence of institutional structures, including market forces, on the use of natural resources. Within a non-equilibrium evolutionary perspective, we highlight the difficulty of choosing a landscape and biodiversity of reference and postulating that it is in equilibrium with a type of social organisation. We conclude by exploring an ‘adaptive management’ approach to the management of the biodiverse landscape studied.     

Kinesin spindle protein (KSP) inhibitors. Part 3: synthesis and evaluation of phenolic 2,4-diaryl-2,5-dihydropyrroles with reduced hERG binding and employment of a phosphate prodrug strategy for aqueous solubility

2,4-Diaryl-2,5-dihydropyrroles have been discovered to be novel, potent and water-soluble inhibitors of KSP, an emerging therapeutic target for the treatment of cancer. A potential concern for these basic KSP inhibitors (1 and 2) was hERG binding that can be minimized by incorporation of a potency-enhancing C2 phenol combined with neutral N1 side chains. Aqueous solubility was restored to these, and other, non-basic inhibitors, through a phosphate prodrug strategy.     

Kinesin spindle protein (KSP) inhibitors. Part 2: the design, synthesis, and characterization of 2,4-diaryl-2,5-dihydropyrrole inhibitors of the mitotic kinesin KSP

The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s<10nM. Ancillary hERG activity was evaluated for this series of inhibitors.     

Kinesin spindle protein (KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of the mitotic kinesin KSP

Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.     

Differential biofilm formation and motility associated with lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia WR-C is capable of forming biofilm on polystyrene and glass. The lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes rmlA, rmlC, and xanB are necessary for biofilm formation and twitching motility. Mutants with mutations in rmlAC and xanB display contrasting biofilm phenotypes on polystyrene and glass and differ in swimming motility.     

Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-beta family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-alpha/beta (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.