CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.     

A functionally divergent hydrogenosomal peptidase with protomitochondrial ancestry

Matrix proteins of mitochondria, hydrogenosomes and mitosomes are typically targeted and translocated into their respective organelles using N-terminal presequences that are subsequently cleaved by a peptidase. Here we characterize a approximately 47 kDa metallopeptidase, from the hydrogenosome-bearing, unicellular eukaryote Trichomonas vaginalis, that contains the active site motif (HXXEHX(76)E) characteristic of the beta subunit of the mitochondrial processing peptidase (MPP) and localizes to hydrogenosomes. The purified recombinant protein, named hydrogenosomal processing peptidase (HPP), is capable of cleaving a hydrogenosomal presequence in vitro, in contrast to MPP which requires both an alpha and beta subunit for activity. T. vaginalis HPP forms an approximately 100 kDa homodimer in vitro and also exists in an approximately 100 kDa complex in vivo. Our phylogenetic analyses support a common origin for HPP and betaMPP and demonstrate that gene duplication gave rise to alphaMPP and betaMPP before the divergence of T. vaginalis and mitochondria-bearing lineages. These data, together with published analyses of MPPs and putative mitosomal processing peptidases, lead us to propose that the length of targeting presequences and the subunit composition of organellar processing peptidases evolved in concert. Specifically, longer mitochondrial presequences may have evolved to require an alpha/beta heterodimer for accurate cleavage, while shorter hydrogenosomal and mitosomal presequences did not.     

YphC and YsxC GTPases assist the maturation of the central protuberance,GTPase associated region and functional core of the 50S ribosomal subunit

YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45S_YphC and 44.5S_YsxC particles. Quantitative mass spectrometry analysis and the 5–6 Å resolution cryo-EM maps of the 45S_YphC and 44.5S_YsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit.     

Surface modification and chemical surface analysis of biomaterials

The chemical composition of the surface layers of synthetic biomaterials used for human medical devices and in biotechnology plays a key role in determining interfacial interactions between biological media (such as protein solutions, cells, tissue) and the synthetic material. Accordingly, considerable research efforts focus on improving the 'biocompatibility' of biomaterials by applying various surface modification and thin film coating approaches. Here we focus on the patterning of surface chemistries, often designed to exercise spatial control over events such as cell attachment and spreading. Secondly, we review recent developments in chemical characterisation of biomaterials surfaces, which is essential both for verifying the success of intended surface modification strategies and for reliable interpretation of observed biological responses. Biomaterials surface analysis by imaging ToF-SIMS and XPS and compositional depth profiling are discussed, as is the emerging complementary technique of Metastable Induced Electron Spectroscopy.     

Effects of S1 Cleavage on the Structure,Surface Export,and Signaling Activity of Human Notch1 and Notch2

Background

Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.

Principal Findings

The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity.

Conclusions/Significance

S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage.     

As old as the hills: Pliocene palaeogeographical processes influence patterns of genetic structure in the widespread,common shrub Banksia sessilis

The impact of Quaternary glaciation on the development of phylogeographic structure in plant species is well documented. In unglaciated landscapes, phylogeographic patterns tend to reflect processes relating to persistence and stochasticity, yet other factors, associated with the palaeogeographical history of the landscape, including geomorphological events, can also have a significant influence. The unglaciated landscape of south‐western Western Australia is an ideal location to observe these ancient drivers of lineage diversification, with tectonic activity associated with the Darling Fault in the late Pliocene attributed to patterns of deep phylogeographic divergence in a widespread tree from this region. Interestingly, other species within this region have not shown this pattern and this palaeogeographical boundary therefore presents an opportunity to examine age and historical distribution of plant species endemic to this region. In this study, we assess patterns of genetic diversity and structure across 28 populations of the widespread shrub Banksia sessilis using three cpDNA markers and nine nuclear microsatellite markers. Sixteen cpDNA haplotypes were identified, comprising two major chloroplast DNA lineages that are estimated to have diverged in the Pliocene, approximately 3.3 million years ago. This timing coincides with major geomorphological processes in the landscape, including the separation of the Darling Plateau from the adjacent Swan Coastal Plain, as well as eustatic changes on the Swan Coastal Plain that are likely to have resulted in the physical isolation of historical plant lineages. Chloroplast lineages were broadly aligned with populations associated with older lateritic soils of the Darling Plateau and Geraldton sandplains or the younger sandy soils associated with the Swan Coastal Plain and Southern Coastline. This structural pattern of lateritic versus non‐lateritic division was not observed in the nuclear microsatellite data that identified three genetic clades that roughly corresponded to populations in the North, South, and Central portions of the distributions.     

Il-13 and IFN-gamma: interactions in lung inflammation

Chronic inflammatory diseases of the lungs, such as asthma, are frequently associated with mixed (Th2 and Th1) T cell responses. We examined the impact of critical Th1 and Th2 cytokines, IFN-gamma and IL-13, on the responses in the lungs. In a mouse model of airway inflammation induced by mixed T cell responses, the number of Th1 (IFN-gamma-positive) cells was found to be negatively correlated with airway hyperreactivity. In these mice, blockade of IL-13 partially inhibited airway hyperreactivity and goblet cell hyperplasia but not inflammation. In contrast, in mice that responded with a polarized Th2 response to the same Ag, blockade of IL-13 inhibited airway hyperreactivity, goblet cell hyperplasia, and airway inflammation. These results indicated that the presence of IFN-gamma would modulate the effects of IL-13 in the lungs. To test this hypothesis, wild-type mice were given recombinant cytokines intranasally. IFN-gamma inhibited IL-13-induced goblet cell hyperplasia and airway eosinophilia. At the same time, IFN-gamma and IL-13 potentiated each other's effects. In the airways of mice given IL-13 and IFN-gamma, levels of IL-6 were increased as well as numbers of NK cells and of CD11c-positive cells expressing MHC class II and high levels of CD86. In conclusion, IFN-gamma has double-sided effects (inhibiting some, potentiating others) on IL-13-induced changes in the lungs. This may be the reason for the ambiguous role of Th1 responses on Th2 response-induced lung injury.     

Mercury in the marine food chain in the Southern Ocean at Macquarie Island: an analysis of a top predator,Patagonian toothfish (<Emphasis Type="Italic">Dissostichus eleginoides</Emphasis>) and a mid-trophic species,the warty squid (<Emphasis Type="Italic">Moroteuthis ingens</Emphasis>)

The characteristics and habitat of the Patagonian toothfish (Dissostichus eleginoides) are typical of fish that accumulate high concentrations of mercury. In this study, mercury determinations were made on samples of muscle tissue from Macquarie Island toothfish and the Southern Ocean deepwater warty squid (Moroteuthis ingens). The analysis of mercury in the biological tissues was made by cold vapour-atomic absorption spectrometry following acid digestion. Performance of the analytical procedure was assessed by analysis of certified reference material (DORM-2, dogfish muscle). Mercury concentrations of 16 Macquarie Island toothfish ranged from 0.12 mg kg^–1 (550 g, 381 mm TL) to 0.59 mg kg^–1 (6,100 g, 823 mm TL), with a mean concentration of 0.33±0.12 mg kg^–1. A significant correlation was found between mercury and either toothfish weight or total length. The fish analysed were juveniles, which suggests that larger individuals would have higher mercury concentrations well exceeding food standard code limits for mercury in fish (typically 0.5 mg kg^–1). Warty squid, also from around Macquarie Island, had a low mean mercury concentration of 0.086 mg kg^–1 in mantle tissue; no significant correlation existed between mercury concentration and either squid mantle length or total weight. It is postulated that the squid have a mechanism, possibly involving the digestive gland, that prevents bioaccumulation of mercury in the mantle, and presumably other body tissues.