Influence of oligomerization state on the structural properties of invasion plasmid antigen B from Shigella flexneri in the presence and absence of phospholipid membranes

Shigella flexneri causes bacillary dysentery, an important cause of mortality among children in the developing world. Shigella secretes effector proteins via its type III secretion system (T3SS) to promote bacterial uptake into human colonic epithelial cells. The T3SS basal body spans the bacterial cell envelope anchoring a surface‐exposed needle. A pentamer of invasion plasmid antigen D lies at the nascent needle tip and invasion plasmid antigen B (IpaB) is recruited into the needle tip complex on exposure to bile salts. From here, IpaB forms a translocon pore in the host cell membrane. Although the mechanism by which IpaB inserts into the membrane is unknown, it was recently shown that recombinant IpaB can exist as either a monomer or tetramer. Both of these forms of IpaB associate with membranes, however, only the tetramer forms pores in liposomes. To reveal differences between these membrane‐binding events, Cys mutations were introduced throughout IpaB, allowing site‐specific fluorescence labeling. Fluorescence quenching was used to determine the influence of oligomerization and/or membrane association on the accessibility of different IpaB regions to small solutes. The data show that the hydrophobic region of tetrameric IpaB is more accessible to solvent relative to the monomer. The hydrophobic region appears to promote membrane interaction for both forms of IpaB, however, more of the hydrophobic region is protected from solvent for the tetramer after membrane association. Limited proteolysis demonstrated that changes in IpaB's oligomeric state may determine the manner by which it associates with phospholipid membranes and the subsequent outcome of this association. Proteins 2014; 82:3013–3022. © 2014 Wiley Periodicals, Inc.     

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N₂ grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.     

LUMBAR SYMPATHECTOMY IN OLDER PATIENTS

Of 43 older patients (aged 65 to 83 years) with arteriosclerosis obliterans treated by lumbar sympathectomy for one or both limbs, 19 had excellent result, 13 had fair result and four had poor result. One died postoperatively and six later. Results were better than prognosticated from response to sympathetic block. Thirty-four patients considered the operation worth while and twelve, after unilateral sympathectomy, requested operation for the other limb also. Twenty-four after operation could walk farther without claudication.     

SketchBio: a scientist’s 3D interface for molecular modeling and animation

Background

Because of the difficulties involved in learning and using 3D modeling and rendering software, many scientists hire programmers or animators to create models and animations. This both slows the discovery process and provides opportunities for miscommunication. Working with multiple collaborators, a tool was developed (based on a set of design goals) to enable them to directly construct models and animations.

Results

SketchBio is presented, a tool that incorporates state-of-the-art bimanual interaction and drop shadows to enable rapid construction of molecular structures and animations. It includes three novel features: crystal-by-example, pose-mode physics, and spring-based layout that accelerate operations common in the formation of molecular models. Design decisions and their consequences are presented, including cases where iterative design was required to produce effective approaches.

Conclusions

The design decisions, novel features, and inclusion of state-of-the-art techniques enabled SketchBio to meet all of its design goals. These features and decisions can be incorporated into existing and new tools to improve their effectiveness.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-334) contains supplementary material, which is available to authorized users.     

Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli

Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2–7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.     

Integrated Human and Ecological Risk Assessment: A Case Study of Tributyltin and Triphenyltin Compounds

Tributyltin and triphenyltin (TBT and TPT) are biocides that have been used to prevent fouling of boats, preserve wood, kill molluscs, and other uses. Due to observed effects on oysters and snails, their use in boat paints has been banned in many nations. However, use on ships and some uses other than as antifouling paints continue. These uses, the relative persistence of these compounds, their tendency to bioaccumulate, and their toxicity cause lingering concerns about risks to humans and non-human organisms. This paper outlines an integrated assessment of TBT and TPT. Based on prior human health and ecological assessments, it suggests that an integrated assessment that recognized common pathways of transport, fate and exposure, and common modes of action would be more efficient and complete than additional independent assessments. The presentation of risks in an integrated manner could also lead to better decisions by defining the various benefits of any management action.     

Opposing Actions of Thrombin and Protease Nexin-1 on Amyloid β-Peptide Toxicity and on Accumulation of Peroxides and Calcium in Hippocampal Neurons

Abstract: Amyloid β-peptide (Aβ) is the principal component of neuritic plaques in the brain in Alzheimer's disease (AD). Recent studies revealed that Aβ can be neurotoxic by a mechanism involving free radical production and loss of cellular ion homeostasis, thus implicating Aβ as a key factor in the pathogenesis of AD. However, other proteins are present in plaques in AD, including the protease thrombin and protease nexin-1 (PN1), a thrombin inhibitor. We therefore tested the hypothesis that thrombin and PN1 modify neuronal vulnerability to Aβ toxicity. In dissociated rat hippocampal cell cultures the toxicity of Aβ was significantly enhanced by coincubation with thrombin, whereas PN1 protected neurons against Aβ toxicity. Aβ induced an increase in levels of intracellular peroxides and calcium. Thrombin enhanced, and PN1 attenuated, the accumulation of peroxides and calcium induced by Aβ. Taken together, these data demonstrate that thrombin and PN1 have opposing effects on neuronal vulnerability to Aβ and suggest that thrombin and PN1 play roles in the pathogenesis of neuronal injury in AD.     

Endogenous 3,4-Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine: LINKS TO MITOCHONDRIALLY DERIVED OXIDATIVE STRESS VIA HYDROXYL RADICAL*

Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3,4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first proteome survey of endogenous site-specific modifications, i.e. DOPA and its further oxidation product dopaquinone in mouse brain and heart tissues. Results from LC-MS/MS analyses included 50 and 14 DOPA-modified tyrosine sites identified from brain and heart, respectively, whereas only a few nitrotyrosine-containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 20 and 12 dopaquinone-modified peptides were observed from brain and heart, respectively; nearly one-fourth of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal binding properties, consistent with metal-catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondrially associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation, suggesting potential disruption of signaling pathways. Collectively, the results suggest that these modifications are linked with mitochondrially derived oxidative stress and may serve as sensitive markers for disease pathologies.Generation of reactive oxygen species (ROS)¹ and reactive nitrogen species is a normal consequence of aerobic metabolism that, in excess, results in oxidative stress that further leads to oxidative modification of proteins, lipids, and DNA, events that may lead to altered cellular function and even cell death (1, 2). Chronic oxidative stress is well recognized as having a central role in disease and is responsible for both direct alteration of biomolecular structure-function and compensatory changes in cellular processes (1–4). It is increasingly recognized that oxidative modifications of proteins can serve as potential biomarkers indicative of the physiological states and changes that occur during disease progression. Thus, the ability to quantitatively measure specific protein oxidation products has the potential to provide the means to monitor the physiological state of a tissue or organism, in particular any progression toward pathology. Given Parkinson disease (PD) as an example, a number of oxidative modifications on proteins pertinent to PD have been identified, further supporting the potential importance of oxidative modifications to disease pathogenesis (5).Many oxidative modifications on specific amino acid residues, such as protein carbonylation (6), cysteine S-nitrosylation (7–9), cysteine oxidation to sulfinic or sulfonic acid (10–12), methionine oxidation (13, 14), and tyrosine nitration (15–21) within complex protein mixtures, have been detected by MS-based proteomics; however, their low abundance levels within complex proteomes often hinder confident identification of these potentially significant modifications (22). For example, tyrosine nitration is a well studied post-translational modification mediated by peroxynitrite (ONOO⁻) or nitrogen dioxide (^·NO₂), which commonly occur in cells during oxidative stress and inflammation; however, only a small number of nitrotyrosine proteins have been identified from a given proteome sample because of insufficient analytical sensitivity and the chance of incorrect peptide assignments (19, 23). With recent advances in high resolution MS that provide high mass measurement accuracy, the ability to confidently identify modified peptides has been significantly enhanced (24).Hydroxyl radical (HO^·) is one of the most reactive and major species generated under aerobic conditions in biological systems (1, 25, 26). Among several HO^·-mediated oxidative modifications, the protein tyrosine modification 3,4-dihydroxyphenylalanine (DOPA) has been reported as a major product and index of HO^· attack on tyrosine residues in proteins (Fig. 1) (27, 28). DOPA is also formed on protein tyrosine residues via controlled enzymatic pathways through enzymes such as tyrosinase or tyrosine hydroxylase (28). Once formed, protein-bound DOPA has the potential to initiate further oxidative reactions through binding and reducing transition metals or through redox cycling between catechol and quinone (dopaquinone) forms (29, 30). Recent studies have suggested that protein-bound DOPA is involved in triggering antioxidant defenses (30) and mediating oxidative damage to DNA (31). Moreover, elevated levels of protein-bound DOPA have been reported in several diseases, including atherosclerosis, cataracts, and myocardial disease, and in PD patients undergoing levodopa therapy (26, 32–36). However, the specific DOPA-modified proteins, which could provide mechanistic knowledge of the progression of these diseases, have not been identified (27, 28). The ability to identify site-specific protein modifications should lead to a better understanding of the role of DOPA modification in disease pathologies as well as new molecular signatures or therapeutic targets for diseases.Open in a separate windowFig. 1.DOPA and dopaquinone formation from tyrosine.Therefore, in this study, we demonstrate the ability to identify site-specific DOPA and dopaquinone (DQ) modifications on protein tyrosine residues in normal mouse brain and heart tissues and their relative stoichiometries that are present in vivo under non-stressed conditions. Such endogenous protein modifications were detected using LC-MS/MS. The results from this global proteomics survey suggests that HO^· in tissues under normal conditions is generated largely from the mitochondria and metal-binding proteins where the resulting DOPA/DQ modifications have the potential to disrupt mitochondrial respiration as well as alter tyrosine phosphorylation signaling pathways such as 14-3-3-mediated signaling in brain tissue.     

Drivers of vegetative dormancy across herbaceous perennial plant species

Vegetative dormancy, that is the temporary absence of aboveground growth for ≥ 1 year, is paradoxical, because plants cannot photosynthesise or flower during dormant periods. We test ecological and evolutionary hypotheses for its widespread persistence. We show that dormancy has evolved numerous times. Most species displaying dormancy exhibit life‐history costs of sprouting, and of dormancy. Short‐lived and mycoheterotrophic species have higher proportions of dormant plants than long‐lived species and species with other nutritional modes. Foliage loss is associated with higher future dormancy levels, suggesting that carbon limitation promotes dormancy. Maximum dormancy duration is shorter under higher precipitation and at higher latitudes, the latter suggesting an important role for competition or herbivory. Study length affects estimates of some demographic parameters. Our results identify life historical and environmental drivers of dormancy. We also highlight the evolutionary importance of the little understood costs of sprouting and growth, latitudinal stress gradients and mixed nutritional modes.