Tubulin polymerization promoting proteins (TPPPs): members of a new family with distinct structures and functions

TPPP/p25 is a brain-specific protein, which induces tubulin polymerization and microtubule (MT) bundling and is enriched in Lewy bodies characteristic of Parkinson's disease [Tirián et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13976-13981]. We identified two human gene sequences, CG1-38 and p25beta, which encoded homologous proteins, that we termed p20 and p18, respectively. These homologous proteins display 60% identity with tubulin polymerization promoting protein/p25 (TPPP/p25); however, the N-terminal segment of TPPP/p25 is missing. They could be clustered into three subfamilies present in mammals and other vertebrates. We cloned, isolated, and characterized the structural and functional properties of the recombinant human proteins at molecular, ultrastructural, and cellular levels using a number of tools. These data revealed that, while p20 behaved as a disorganized protein similarly to TPPP/p25, which was described as a flexible and inherently dynamic protein with a long unstructured N-terminal tail, p18 was featured in more ordered fashion. TPPP/p25 and p20 specifically attached to MTs causing MT bundling both in vitro and in vivo; p18 protein did not cross-link MTs, and it distributed homogeneously within the cytosol of the transfected HeLa cells. These data indicate that the two shorter homologues display distinct structural features that determine their associations to MTs. The properties of p20 resemble TPPP/p25. The bundling activity of these two proteins results in the stabilization of the microtubular network, which is likely related to their physiological functions.     

Recovery of native grass biodiversity by sowing on former croplands: Is weed suppression a feasible goal for grassland restoration?

Grassland restoration on former croplands offers good opportunity to mitigate the loss of grassland biodiversity. Weed suppression can be another benefit, which becomes increasingly important because of the high recent rate of abandonment of arable lands in Central and Eastern Europe. Our aim was to evaluate the usefulness of sowing two low-diversity seed mixtures followed by annual mowing, a frequently used restoration technique, in weed suppression. We found that rapidly forming cover of sown grasses effectively suppressed short-lived weeds and their germination except in the first year. The detected dense seed bank of short-lived weeds points out the possibility and threat of later weed infestation. In the short run perennial weeds cannot be suppressed easily by sowing and annual mowing. We found that the effectiveness of seed sowing followed by mowing in weed suppression can be different on sites with different history or seed mixture. Rapidly establishing perennial weeds, such as Agropyron species were only detected in former alfalfa fields; Cirsium arvense was found in former cereal and sunflower fields but not in former alfalfa fields. We found that the rate of weed suppression and success was influenced by the seed mixtures used. In several alkali restorations the high proportion of perennial weeds was detected in year 3. In loess restorations, much lower scores were typical. This was likely caused by the different seed mixture used. The loess seed mixture contained seeds of a clonally spreading tall-grass, Bromus inermis, which could compete more effectively with clonally spreading weeds, than could short grass species with or without tussock forming. Our findings indicate that post-restoration management require carefully designed actions that are fine-tuned addressing specific threats at the site level.     

Down feather morphology reflects adaptation to habitat and thermal conditions across the avian phylogeny

Down feathers are the first feather types that appear in both the phylogenetic and the ontogenetic history of birds. Although it is widely acknowledged that the primary function of downy elements is insulation, little is known about the interspecific variability in the structural morphology of these feathers, and the environmental factors that have influenced their evolution. Here, we collected samples of down and afterfeathers from 156 bird species and measured key morphological characters that define the insulatory properties of the downy layer. We then tested if habitat and climatic conditions could explain the observed between-species variation in down feather structure. We show that habitat has a very strong and clearly defined effect on down feather morphology. Feather size, barbule length and nodus density all decreased from terrestrial toward aquatic birds, with riparian species exhibiting intermediate characters. Wintering climate, expressed as windchill (a combined measure of the ambient temperature and wind speed) had limited effects on down morphology, colder climate only being associated with higher nodus density in dorsal down feathers. Overall, an aquatic lifestyle selects for a denser plumulaceous layer, while the effect of harsh wintering conditions on downy structures appear limited. These results provide key evidence of adaptations to habitat at the level of the downy layer, both on the scale of macro- and micro-elements of the plumage. Moreover, they reveal characters of convergent evolution in the avian plumage and mammalian fur, that match the varying needs of insulation in terrestrial and aquatic modes of life.     

Phytochemical and pharmacological investigation of <Emphasis Type="Italic">Spiraea chamaedryfolia</Emphasis>: a contribution to the chemotaxonomy of <Emphasis Type="Italic">Spiraea</Emphasis> genus

Objective

Diterpene alkaloids are secondary plant metabolites and chemotaxonomical markers with a strong biological activity. These compounds are characteristic for the Ranunculaceae family, while their occurrence in other taxa is rare. Several species of the Spiraea genus (Rosaceae) are examples of this rarity. Screening Spiraea species for alkaloid content is a chemotaxonomical approach to clarify the classification and phylogeny of the genus. Novel pharmacological findings make further investigations of Spiraea diterpene alkaloids promising.

Results

Seven Spiraea species were screened for diterpene alkaloids. Phytochemical and pharmacological investigations were performed on Spiraea chamaedryfolia, the species found to contain diterpene alkaloids. Its alkaloid-rich fractions were found to exert a remarkable xanthine-oxidase inhibitory activity and a moderate antibacterial activity. The alkaloid distribution within the root was clarified by microscopic techniques.

     

Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase

dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution. Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site. Results provide an understanding for the catalytic role of conserved residues in dUTPases.     

Bacterial evolution of antibiotic hypersensitivity

The evolution of resistance to a single antibiotic is frequently accompanied by increased resistance to multiple other antimicrobial agents. In sharp contrast, very little is known about the frequency and mechanisms underlying collateral sensitivity. In this case, genetic adaptation under antibiotic stress yields enhanced sensitivity to other antibiotics. Using large‐scale laboratory evolutionary experiments with Escherichia coli, we demonstrate that collateral sensitivity occurs frequently during the evolution of antibiotic resistance. Specifically, populations adapted to aminoglycosides have an especially low fitness in the presence of several other antibiotics. Whole‐genome sequencing of laboratory‐evolved strains revealed multiple mechanisms underlying aminoglycoside resistance, including a reduction in the proton‐motive force (PMF) across the inner membrane. We propose that as a side effect, these mutations diminish the activity of PMF‐dependent major efflux pumps (including the AcrAB transporter), leading to hypersensitivity to several other antibiotics. More generally, our work offers an insight into the mechanisms that drive the evolution of negative trade‐offs under antibiotic selection.     

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL

We found that heme-binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub-threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme-binding properties SOUL promotes necrotic cell death by inducing mPT.