Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.     

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.     

Introducing Intron Locus cox1i624 for Phylogenetic Analyses in Bryophytes: On the Issue of Takakia as Sister Genus to All Other Extant Mosses

Liverworts are well supported as the sister group to all other land plants (embryophytes) by molecular data. Observations strongly supporting this earliest dichotomy in embryophyte evolution are the strikingly different introns occurring in the mitochondrial DNAs of liverworts versus non-liverwort embryophytes (NLE), including the mosses. A final conclusion on the most basal lineages of mosses, for which genera such as Sphagnum and Takakia are the most likely candidates, is lacking. We have now investigated cox1i624, a mitochondrial group I intron conserved between the moss Physcomitrella patens and the liverwort Marchantia polymorpha. Focusing on a sampling of liverwort and moss genera, which had previously been identified as early branching taxa in their respective clades, we find that group I intron cox1i624 is universally conserved in all 33 mosses and 11 liverworts investigated. The group I intron core secondary structure is well conserved between the two ancient land plant clades. However, whereas dramatic size reductions are seen in the moss phylogeny, exactly the opposite is observed for liverworts. The cox1i624g1 locus was used for phylogenetic tree reconstruction also in combination with data sets of nad5i753g1 as well as chloroplast loci rbcL and rps4. The phylogenetic analyses revealed (i) very good support for the Treubiopsida as sister clade to all other liverworts, (ii) a sister group relationship of the nematodontous Tetraphidopsida and Polytrichopsida and (iii) two rivalling hypotheses about the basal-most moss genus with mitochondrial loci suggesting an isolated Takakia as sister to all other mosses and chloroplast loci indicating a Takakia–Sphagnum clade.     

Biological roles of APP in the epidermis

The amyloid precursor protein (APP) was initially detected in cells of the central nervous system where it is considered to be involved in the pathogenesis of Alzheimer's disease. However, APP is also found in peripheral organs with exceptionally strong expression in the mammalian epidermis where it fulfils a variety of distinct biological roles. Full length APP appears to facilitate keratinocyte adhesion due to its ability to interact with the extracellular matrix. The C-terminus of APP also serves as adapter protein for binding the motor protein kinesin thereby mediating the centripetal transport of melanosomes in epidermal melanocytes. By the action of alpha-secretase sAPPalpha, the soluble N-terminal portion of APP, is released. sAPPalpha has been shown to be a potent epidermal growth factor thus stimulating proliferation and migration of keratinocytes as well as the exocytic release of melanin by melanocytes. The release of sAPPalpha can be almost completely blocked by inhibiting alpha-secretase with hydroxamic acid-based zinc metalloproteinase inhibitors. In hyperproliferative keratinocytes from psoriatic skin this inhibition results in normalized growth.     

Temperature and photoperiod affect stress resistance traits in Drosophila melanogaster

The long‐term survival of species and populations depends on their ability to adjust phenotypic values to environmental conditions. In particular, the capability of dealing with environmental stress to buffer detrimental effects on fitness is considered to be of pivotal importance. Resistance traits are readily modulated by a wide range of environmental factors. In the present study, Drosophila melanogaster Meigen is used to investigate plastic responses to temperature and photoperiod in stress resistance traits. The results reveal that stress resistance traits (cold, heat, starvation and desiccation resistance) are affected by the factors temperature and sex predominantly. Cooler temperatures compared with warmer temperatures increase cold tolerance, desiccation and starvation resistance, whereas they reduce heat tolerance. Except for heat resistance, females are more stress‐resistant than males. Stress resistance traits are also affected by photoperiod. Shorter photoperiods decrease cold tolerance, whereas longer photoperiods enhance desiccation resistance. Overall, thermal effects are pervasive throughout all measured resistance traits, whereas photoperiodic effects are of limited importance in the directly developing (i.e. nondiapausing) flies used here, suggesting that pronounced photoperiodic effects on stress resistance traits may be largely limited to, and triggered by, diapause‐inducing effects.     

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

Neutral mechanisms and niche differentiation in steady-state insular microbial communities revealed by single cell analysis

In completely insular microbial communities, evolution of community structure cannot be shaped by the immigration of new members. In addition, when those communities are run in steady state, the influence of environmental factors on their assembly is reduced. Therefore, one would expect similar community structures under steady-state conditions. Yet, in parallel setups, variability does occur. To reveal ecological mechanisms behind this phenomenon, five parallel reactors were studied at the single-cell level for about 100 generations and community structure variations were quantified by ecological measures. Whether community variability can be controlled was tested by implementing soft temperature stressors as potential synchronizers. The low slope of the lognormal rank-order abundance curves indicated a predominance of neutral mechanisms, i.e., where species identity plays no role. Variations in abundance ranks of subcommunities and increase in inter-community pairwise β-diversity over time support this. Niche differentiation was also observed, as indicated by steeper geometric-like rank-order abundance curves and increased numbers of correlations between abiotic and biotic parameters during initial adaptation and after disturbances. Still, neutral forces dominated community assembly. Our findings suggest that complex microbial communities in insular steady-state environments can be difficult to synchronize and maintained in their original or desired structure, as they are non-equilibrium systems.     

The fine structure of protonephridia in Gnathostomulida and their comparison within Bilateria

Summary The fine structure of the protonephridia of Haplognathia rosea (Filospermoidea) and Gnathostomula paradoxa (Bursovaginoidea) is described. Each protonephridium consists of three different cells: (1) a monociliated terminal cell which constitutes the filtration area, (2) a nonciliated canal cell showing a special protonephridial outlet system, and (3) an intraepidermal cell — the nephroporus cell — constituting the nephroporus. The protonephridia are arranged serially. There is no canal system connecting the protonephridial units.Protonephridial characters in other Bilateria are considered. The pattern of characters in the protonephridia in the last common gnathostomulid stem species and presumed apomorphies in the protonephridia of the Gnathostomulida investigated are discussed.Abbreviations used in figures ac acessory centriole - AC additional epidermal cell - bb basal body - bl basal lamina - bm bundle of microvilli - c cilium - cc cilium duct cell - cd cilium duct - cr ciliary rootlet - crs structures resembling ciliary rootlets - di diplosome - ds desmosome - dy dictyosome - f filtration area - g granules - m mitochondrium - mv microvillus - n nucleus - NC nephroporus cell - np nephroporus - oc outlet canal - TC terminal cell - tl tubules of lacunar system