Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.     

Bleb-driven chemotaxis of Dictyostelium cells

Blebs and F-actin–driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.     

Ceramide triggers intracellular calcium release via the IP3 receptor in Xenopus laevis oocytes

Ceramide, a product of sphingomyelin turnover, is a lipid secondmessenger that mediates diverse signaling pathways, including thoseleading to cell cycle arrest and differentiation. The mechanism(s) bywhich ceramide signals downstream events have not been fully elucidated. Here we show that, in Xenopuslaevis oocytes, ceramide-induced maturation isassociated with the release of intracellular calcium stores. Ceramidecaused a dose-dependent elevation in the second messenger inositol1,4,5-trisphosphate (IP₃) viaactivation of G_q/11 andphospholipase C-X. Elevation ofIP₃, in turn, activated theIP₃ receptor calcium releasechannel on the endoplasmic reticulum, resulting in a rise incytoplasmic calcium. Thus our study demonstrates that cross talkbetween the ceramide and phosphoinositide signaling pathways modulatesintracellular calcium homeostasis.

     

Temporal area approach for distributional data in biogeography

A structural approach to temporality in distributional data for use in palaeobiogeography is described herein. Pre-established areas in the distributional data matrix are split temporally, allowing a single geographical space to have multiple iterations [e.g. Area A (Lower Devonian), Area A (Middle Devonian)]. The resulting temporal matrix will allow the representation and capture of any differing relationships through time. Designed primarily for Parsimony Analysis of Endemicity (PAE) and biotic similarity analyses, this approach simply structures distributional data within a temporal partition, meaning that numerical methods can be used to assess relationships between areas to find a branching diagram. Created through the application of the temporal matrix to a given analysis, Temporal Area Approach (TAAp) is a structural approach that facilitates exploration of the data rather than being a hypothesis-driven model following analysis. Understanding the behaviour of non-phylogenetic palaeobiogeographical data and reducing the prevalence of temporal artefacts will lead to more robust area classifications.     

Neutral sphingomyelinase 2 deficiency is associated with lung anomalies similar to emphysema

Neutral sphingomyelinase 2 (nSMase2) upregulation was recently demonstrated to serve as a molecular link between smoke inhalation and emphysematous changes in lungs. Here we report that nSMase2 deficit impairs lung development in mice. We have shown previously that fragilitas ossium (fro) mice carry a mutation in the Smpd3 gene, rendering nSMase2 catalytically inactive. Analysis of lung phenotype revealed that fro mice have abnormally enlarged alveoli and increased compliance of the respiratory system, similar to morphological and functional manifestations of emphysema. Analysis of sphingolipid content in fro lungs revealed a decreased level of C14:0 ceramide but no significant alterations in the levels of sphingosine or sphingosine-1-phosphate. Altogether, our data suggest that nSMase2 activity and ceramide level are critical for lung development and function. Based on our data, ceramide can no longer be viewed as a lipid solely detrimental to lung function.

     

Sample preparation and analytical strategies for large-scale phosphoproteomics experiments

Reversible protein phosphorylation is an important post-translational modification that controls a wide range of protein functions including enzyme activity, subcellular localisation, protein degradation, intra- and inter-molecular protein interactions. Significant advances in both phosphopeptide enrichment methods and sensitive mass spectrometry instrumentation have been achieved over the past decade to facilitate the large-scale identification of protein phosphorylation in humans and different animal and microbial model systems. While mass spectrometry provides the ability to identify thousands of phosphorylation sites in a single experiment, the further understanding of the functional significance of this modification on protein substrates requires detailed information on the changes in phosphorylation stoichiometry and protein abundance across experimental paradigms. This review presents different sample preparation methods and analytical strategies used in mass spectrometry-based phosphoproteomics to profile protein phosphorylation and unravel the regulation of this modification on protein function.     

Evolution of Pan-Genomes of Escherichia coli,Shigella spp., and Salmonella enterica

Multiple sequencing of genomes belonging to a bacterial species allows one to analyze and compare statistics and dynamics of the gene complements of species, their pan-genomes. Here, we analyzed multiple genomes of Escherichia coli, Shigella spp., and Salmonella enterica. We demonstrate that the distribution of the number of genomes harboring a gene is well approximated by a sum of two power functions, describing frequent genes (present in many strains) and rare genes (present in few strains). The virtual absence of Shigella-specific genes not present in E. coli genomes confirms previous observations that Shigella is not an independent genus. While the pan-genome size is increasing with each new strain, the number of genes present in a fixed fraction of strains stabilizes quickly. For instance, slightly fewer than 4,000 genes are present in at least half of any group of E. coli genomes. Comparison of S. enterica and E. coli pan-genomes revealed the existence of a common periphery, that is, genes present in some but not all strains of both species. Analysis of phylogenetic trees demonstrates that rare genes from the periphery likely evolve under horizontal transfer, whereas frequent periphery genes may have been inherited from the periphery genome of the common ancestor.     

Long-term H<Subscript>2</Subscript> photoproduction from starch by co-culture of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in a repeated batch process

Objectives

To prove the possibility of efficient starch photofermentation in co-culture of heterotrophic and phototrophic bacteria over prolonged period.

Results

Repeated batch photofermentation of starch was demonstrated in co-culture Clostridium butyricum and Rhodobacter sphaeroides under microaerobic conditions. It continued 15 months without addition of new inoculum or pH regulation when using 4–5 g starch l^?1 and 0.04 g yeast extract l^?1. The complete degradation of starch without volatile fatty acids accumulation was shown in this co-culture. The average H₂ yield of 5.2 mol/mol glucose was much higher than that in Clostridium monoculture. The species composition of co-culture was studied by q-PCR assay. The concentration of Clostridium cells in prolonged co-culture was lower than in monoculture and even in a single batch co-culture. This means that Clostridia growth was significantly limited whereas starch hydrolysis still took place.

Conclusion

The prolonged repeated batch photofermentation of starch by co-culture C. butyricum and R. sphaeroides provided efficient H₂ production without accumulation of organic acids under conditions of Clostridia limitation.