Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a K_d of ca. 10⁻⁵ M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI''s interaction with a partly-folded protein, and the first to analyze this folding catalyst''s changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility.     

Strain Diversity of CTX-M-Producing Enterobacteriaceae in Individual Pigs: Insights into the Dynamics of Shedding during the Production Cycle

The aim of this study was to evaluate the population dynamics of CTX-M-producing Enterobacteriaceae in individual pigs on a farm positive for CTX-M-14-producing Escherichia coli. Fecal samples were collected once around the farrowing time from five sows and four times along the production cycle from two of their respective offspring. Multiple colonies per sample were isolated on cefotaxime-supplemented MacConkey agar with or without prior enrichment, resulting in 98 isolates identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry and tested for bla_CTX-M. CTX-M-positive isolates (n = 86) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harboring bla_CTX-M were characterized in 22 representative isolates by replicon typing and restriction fragment length polymorphism. Based on the PFGE results, all individuals shed unrelated CTX-M-14-producing E. coli strains during the course of life. Concomitant shedding of CTX-M-2/97-producing Proteus mirabilis or Providencia rettgeri was observed in two sows and two offspring. At least two genetically unrelated CTX-M-producing E. coli strains were isolated from approximately one-fourth of the samples, with remarkable differences between isolates obtained by enrichment and direct plating. A clear decrease in strain diversity was observed after weaning. Dissemination of bla_CTX-M-14 within the farm was attributed to horizontal transfer of an IncK plasmid that did not carry additional resistance genes and persisted in the absence of antimicrobial selective pressure. Assessment of strain diversity was shown to be influenced by the production stage from which samples were collected, as well as by the isolation method, providing useful information for the design and interpretation of future epidemiological studies of CTX-M-producing Enterobacteriaceae in pig farms.     

Evolution of cave suspension feeding in Protodrilidae (Annelida)

Protodrilidae belongs in a lineage that until now entirely consisted of deposit‐feeding, highly adapted interstitial annelids. Except for a pair of anterior palps, all protodrilids lack appendages, parapodia and chaetae; and have slender bodies adapted to glide between the sand grains by ciliary motion. The first exception to these characteristics is Megadrilus pelagicus n. sp. inhabiting the water column of the anchialine La Corona cave system in Lanzarote. Its morphology and evolutionary history are here investigated by combining observations from in vivo video recordings and advanced microscopy with phylogenetic analyses. Our studies revealed a unique pelagic, suspension feeding behaviour attained by its long ciliated palps in combination with an autopomorphic dorsal ciliated keel and several longitudinal and transverse ciliary bands. Phylogenetic analyses recovered Megadrilus pelagicus n. sp. nested within Protodrilidae indicating that its unique traits are derived within the family. These traits are traced in the tree topologies in correlation to cave colonization. The evolution of these traits can be functionally explained by the different demands of a pelagic suspension feeding strategy compared to the ancestral deposit‐feeding guild of the family. The origin of this suspension feeding strategy was presumably favoured by the partial isolation of the anchialine ecosystem, connected to the sea only through the highly porous volcanic subterranean bedrock. This crevicular connection limits the amount of predators and turbulence in the cave, but allows continuous water flow into the system carrying organic particles, which is the main source of food when photosynthetic primary production does not occur and sedimentation is limited. These conditions may select for pelagic suspension feeding as the most feasible life‐strategy in anchialine caves, which the dominance of pelagic, suspension feeding crustaceans and annelids in anchialine cave assemblages may also reflect. For species of ancestrally deposit‐feeding lineages entering the cave system, such as the annelid families Protodrilidae and Nerillidae, an adaptive‐shift from interstitial to crevicular habitats seemingly correlates with dramatic morphological changes and speciation. The dramatic changes observed in these primarily interstitial lineages compared to their relatives, point to alternative adaptive evolutionary pathways related to ecological fitness contrary to the previously proposed theories focusing on geological or stochastic processes.     

Psychopathology in 7‐year‐old children with familial high risk of developing schizophrenia spectrum psychosis or bipolar disorder – The Danish High Risk and Resilience Study ‐ VIA 7, a population‐based cohort study

This study aimed to compare the psychopathological profiles of children at familial high risk of schizophrenia spectrum psychosis (FHR‐SZ) or bipolar disorder (FHR‐BP) with population‐based controls. We used Danish nationwide registers to retrieve a cohort of 522 seven‐year‐old children of parents with schizophrenia spectrum psychosis (N=202), bipolar disorder (N=120) or none of these disorders (N=200). Psychopathology was assessed by reports from multiple informants, including children, parents and teachers. Lifetime DSM‐IV diagnoses were ascertained by blinded raters through the Schedule for Affective Disorders and Schizophrenia for School‐Age Children. The dimensional assessment of psychopathology was performed by the Child Behavior Checklist, the Teacher's Report Form, a modified version of the ADHD‐Rating Scale, the Test Observation Form, and the State‐Trait Anxiety Inventory for Children. Current level of functioning was evaluated using the Children's Global Assessment Scale (CGAS). The prevalence of lifetime psychiatric diagnoses was significantly higher in both FHR‐SZ children (38.7%, odds ratio, OR=3.5, 95% confidence interval, CI: 2.2‐5.7, p < 0.001) and FHR‐BP children (35.6%, OR=3.1, 95% CI: 1.8‐5.3, p < 0.001) compared with controls (15.2%). FHR‐SZ children displayed significantly more dimensional psychopathology on all scales and subscales compared with controls except for the Anxious subscale of the Test Observation Form. FHR‐BP children showed higher levels of dimensional psychopathology on several scales and subscales compared with controls, but lower levels compared with FHR‐SZ children. Level of functioning was lower in both FHR‐SZ children (CGAS mean score = 68.2; 95% CI: 66.3‐70.2, p < 0.0001) and FHR‐BP children (73.7; 95% CI: 71.2‐76.3, p < 0.05) compared with controls (77.9; 95% CI: 75.9‐79.9). In conclusion, already at the age of seven, FHR‐SZ and FHR‐BP children show a higher prevalence of a broad spectrum of categorical and dimensional psychopathology compared with controls. These results emphasize the need for developing early intervention strategies towards this vulnerable group of children.     

Unique Substrate Specificity of SplE Serine Protease from Staphylococcus aureus

1. Download high-res image (93KB)
2. Download full-size image

     

High‐throughput metabarcoding of eukaryotic diversity for environmental monitoring of offshore oil‐drilling activities

As global exploitation of available resources increases, operations extend towards sensitive and previously protected ecosystems. It is important to monitor such areas in order to detect, understand and remediate environmental responses to stressors. The natural heterogeneity and complexity of communities means that accurate monitoring requires high resolution, both temporally and spatially, as well as more complete assessments of taxa. Increased resolution and taxonomic coverage is economically challenging using current microscopy‐based monitoring practices. Alternatively, DNA sequencing‐based methods have been suggested for cost‐efficient monitoring, offering additional insights into ecosystem function and disturbance. Here, we applied DNA metabarcoding of eukaryotic communities in marine sediments, in areas of offshore drilling on the Norwegian continental shelf. Forty‐five samples, collected from seven drilling sites in the Troll/Oseberg region, were assessed, using the small subunit ribosomal RNA gene as a taxonomic marker. In agreement with results based on classical morphology‐based monitoring, we were able to identify changes in sediment communities surrounding oil platforms. In addition to overall changes in community structure, we identified several potential indicator taxa, responding to pollutants associated with drilling fluids. These included the metazoan orders Macrodasyida, Macrostomida and Ceriantharia, as well as several ciliates and other protist taxa, typically not targeted by environmental monitoring programmes. Analysis of a co‐occurrence network to study the distribution of taxa across samples provided a framework for better understanding the impact of anthropogenic activities on the benthic food web, generating novel, testable hypotheses of trophic interactions structuring benthic communities.     

Is Diurodrilus an annelid?

Interstitial marine meiofaunal worms of the genus Diurodrilus have always been considered part of Annelida, either as basal or derived, though generally with reference to Dinophilidae. New evidence shows that Diurodrilus has a unique anatomy, and lacks key annelid features, possibly even segmentation. We assessed the systematic position of Diurodrilus among other protostome animals via light microscopy, confocal laser scanning microscopy, and transmission electron microscopy studies of anatomy, focusing on musculature, the nervous system, as well as molecular sequence data. We show that there is little morphological or molecular evidence to support a relationship with Dinophilidae or any other annelids. Diurodrilus has some similarities to Micrognathozoa, though the latter shows complex jaws. On the basis of the configuration of the nervous system and the cuticle we regard Diurodrilus to belong to Spiralia, possibly close to Annelida; however, until further evidence is acquired it should be regarded as incertae sedis in this large animal clade. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Substrate specificity of Staphylococcus aureus cysteine proteases--Staphopains A, B and C

Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2–Gly↓Ala(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.