Conserved substrate binding by chaperones in the bacterial periplasm and the mitochondrial intermembrane space

Mitochondria were derived from intracellular bacteria and the mitochondrial intermembrane space is topologically equivalent to the bacterial periplasm. Both compartments contain ATP-independent chaperones involved in the transport of hydrophobic membrane proteins. The mitochondrial TIM (translocase of the mitochondrial inner membrane) 10 complex and the periplasmic chaperone SurA were examined in terms of evolutionary relation, structural similarity, substrate binding specificity and their function in transporting polypeptides for insertion into membranes. The two chaperones are evolutionarily unrelated; structurally, they are also distinct both in their characteristics, as determined by SAXS (small-angle X-ray scattering), and in pairwise structural comparison using the distance matrix alignment (DALILite server). Despite their structural differences, SurA and the TIM10 complex share a common binding specificity in Pepscan assays of substrate proteins. Comprehensive analysis of the binding on a total of 1407 immobilized 13-mer peptides revealed that the TIM10 complex, like SurA, does not bind hydrophobic peptides generally, but that both chaperones display selectivity for peptides rich in aromatic residues and with net positive charge. This common binding specificity was not sufficient for SurA to completely replace TIM10 in yeast cells in vivo. In yeast cells lacking TIM10, when SurA is targeted to the intermembrane space of mitochondria, it binds translocating substrate proteins, but fails to completely transfer the substrate to the translocase in the mitochondrial inner membrane. We suggest that SurA was incapable of presenting substrates effectively to the primitive TOM (translocase of the mitochondrial outer membrane) and TIM complexes in early mitochondria, and was replaced by the more effective small Tim chaperone.     

Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F.

PNGase F is a widely used deglycosidase, secreted in small amounts by the gram-negative bacterium Flavobacterium meningosepticum. We have designed a T7 promoter-based Escherichia coli expression system to provide a high-yield source of recombinant enzyme. When expressed intracellularly, the enzyme was produced in a largely insoluble state. However, when expressed as a fusion with the leader sequence from the ompA gene, hexahistidine-tagged PNGase F was efficiently processed and exported to the E. coli periplasm. Single-step purification using immobilized metal affinity chromatography yielded 8 mg of pure enzyme per liter of culture, which is fully active on a range of protein and peptide substrates.     

Infection of Anastrepha ludens following soil applications of Heterorhabditis bacteriophora in a mango orchard

To determine the efficacy of Heterorhabditis bacteriophora Poinar (Nematoda: Heterorhabditidae) for control of Anastrepha ludens (Loew) (Diptera: Tephritidae), field experiments were performed in a mango orchard with soil temperatures of 24–29 °C. The density of third‐instar A. ludens (50–500 larvae per plot) released into 0.25 m² wood‐framed experimental plots containing soil (16% wt/wt moisture) previously treated with 125 infective juveniles per square centimetre soil surface did not significantly influence the prevalence of infection by H. bacteriophora. In subsequent experiments, the percentages of infection of fly pupae were positively correlated with the concentration of infective stages applied to soil plots. The highest average percentage of infection (74% at 250 infective juveniles per square centimetre) was observed when fly larvae were released simultaneously onto soil, compared to larvae that emerged from laboratory‐infested mangoes over a period of 8 days (52% infection at 500 infective juveniles per square centimetre). Double applications of infective juveniles at an interval of 4 days did not greatly improve the prevalence of infection (～10% higher) compared to single applications. Between 9 and 15% of larvae that remained within infested mangoes became infected by nematodes, irrespective of the concentration of nematodes applied to each experimental plot. We conclude that effective control of A. ludens requires very high densities of H. bacteriophora. The successful use of this nematode for biocontrol of A. ludens will depend on identifying ways of overcoming the fly's ability to avoid infection.     

A Plains‐wanderer (Pedionomidae) that did not wander plains: a new species from the Oligocene of South Australia

The remarkable fauna of Australia evolved in isolation from other landmasses for millions of years, yet understanding the evolutionary history of endemic avian lineages on the continent is confounded by the ability of birds to disperse over geographical barriers even after vicariance events. The Plains‐wanderer Pedionomus torquatus (Charadriiformes) is an enigmatic, predominantly sedentary, quail‐like bird that occurs exclusively in sparse native grasslands of southeastern Australia. It is the only known species of its family (Pedionomidae), and its closest relatives are the South American seedsnipes (Thinocoridae). Here we describe a further representative of this lineage, Oligonomus milleri gen. et sp. nov., from the Late Oligocene of South Australia (26–24 Ma), which pre‐dates the earliest record of P. torquatus by c. 22 Ma and attests to the presence of this lineage during Australia's period of isolation (50–15 Ma). Based on the morphology of the coracoid and the palynological record, we propose that O. milleri and P. torquatus were ecologically disparate taxa and that, similar to coeval marsupials, O. milleri inhabited well‐wooded habitats, suggesting that the preference for grassland in the extant P. torquatus and thinocorids is likely to be convergent and not ancestral. The speciation event leading to the evolution of the extant Plains‐wanderer was probably triggered by the spread of grasslands across Australia in the Late Miocene–Pliocene, which this record pre‐dates. The presence of a pedionomid in the Late Oligocene of Australia strengthens the hypothesis of a Gondwanan divergence of the lineages giving rise to Thinocoridae and Pedionomidae.     

Effect of Different Interferonα2 Preparations on IP10 and ET-1 Release from Human Lung Cells

Background

Alfa-interferons (IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b) are effective treatments for chronic hepatitis C infection. However, their usage has been associated with a variety of adverse events, including interstitial pneumonitis and pulmonary arterial hypertension. Although rare, these adverse events can be severe and potentially life-threatening, emphasizing the need for simple biomarkers of IFN-induced lung toxicity.

Methods

Human lung microvascular endothelial cells (HLMVEC), human pulmonary artery smooth muscle (HPASM) cells and A549 cells were grown under standard conditions and plated into 96- or 6-well plates. Cells were stimulated with various concentrations of different IFNs in hydrocortisone-free medium. After 24 and 48 hours, IP10 and ET-1 were measured by ELISA in conditioned medium. In a second set of experiments, cells were pre-treated with tumour necrosis factor-α (TNF-α) (10 ng/mL).

Results

IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b, but not IFNλ, induced IP10 (CXCL10) release and increased IP10 gene induction in HLMVEC. In addition, all four IFNα preparations induced IP10 release from HPASM cells and A549 cells pre-treated with TNFα. In each of these cell types, 40KDa-PEGIFNα2a was significantly less active than the native forms of IFNα2a, IFNα2b or 12KDa-PEGIFNα2b. Similarly, IFNα2a, IFNα2b and 12KDa-PEGIFNα2b, but not 40KDa-PEGIFNα2a, induced endothelin (ET)-1 release from HPASM cells.

Conclusions

Consistent with other interstitial pulmonary diseases, both IP10 and ET1 may serve as markers to monitor IFN-induced lung toxicity in patients. In addition, both markers may also serve to help characterize the risk associated with IFNα preparations to induce lung toxicity.     

On estimating the precision of stable isotope ratios in processed tree-rings

Stable isotope dendrochronology is a well-developed field of research, but improvements to methodologies are on-going. We propose an improved method for estimating the precision of stable isotope ratios (δ) of tree-ring samples that are processed from whole wood to various end products such as cellulose-nitrate, α-cellulose, or cellulose intermediates. The status quo method for estimating the δ precision of organic solids is to characterise the long-term 2-sigma range of δ values for a ready-made Quality Assurance (QA) standard that is included in each analysis run of samples. While the status quo method is appropriate for characterising analytical uncertainties associated with the mass spectrometer, combustion or pyrolysis system, and analyte specifics, it does not reflect uncertainties associated with sample processing from inadvertent and unrealised operator error (e.g., contamination by airborne particles, incomplete chemical processing, sample storage issues, and other unforeseen errors), although such errors would probably be rare with an experienced operator. The proposed method improves upon the status quo method as it respects the Identical Treatment principle by subjecting QA standards to the same processing steps that samples undergo. As such, analytical uncertainties associated with sample processing would be integrated into the QA standard's δ value and precision estimate. In effect, the proposed method is a system to monitor inter-batch reproducibility and, by the same token, can be used to identify batches that were potentially compromised during processing. A pilot study example is used to demonstrate the proposed method for δ¹⁸O analysis of α-cellulose samples.     

Inference of Unexpected Genetic Relatedness among Individuals in HapMap Phase III

The International Haplotype Map Project (HapMap) has provided an essential database for studies of human population genetics and genome-wide association. Phases I and II of the HapMap project generated genotype data across ∼3 million SNP loci in 270 individuals representing four populations. Phase III provides dense genotype data on ∼1.5 million SNPs, generated by Illumina and Affymetrix platforms in a larger set of individuals. Release 3 of phase III of the HapMap contains 1397 individuals from 11 populations, including 250 of the original 270 phase I and phase II individuals and 1147 additional individuals. Although some known relationships among the phase III individuals have been described in the data release, the genotype data that are currently available provide an opportunity to empirically ascertain previously unknown relationships. We performed a systematic analysis of genetic relatedness and were able not only to confirm the reported relationships, but also to detect numerous additional, previously unidentified pairs of close relatives in the HapMap sample. The inferred relative pairs make it possible to propose standardized subsets of unrelated individuals for use in future studies in which relatedness needs to be clearly defined.     

Chemistry and Pharmacology of Gynostemma pentaphyllum

In traditional Chinese medicine, Gynostemma pentaphyllum (Thunb.) Makino is a herbal drug of extreme versatility and has been extensively researched in China. The dammarane saponins isolated from Gynostemma pentaphyllum, namely gypenosides or gynosaponins, are believed to be the active components responsible for its various biological activities and reported clinical effects. This review attempts to encompass the available literature on Gynostemma pentaphyllum, from its cultivation to the isolation of its chemical entities and a summary of its diverse pharmacological properties attributed to its gypenoside content. Other aspects such as toxicology and pharmacokinetics are also discussed. In vitro and in vivo evidence suggests that Gynostemma pentaphyllum may complement the popular herbal medicine, Panax ginseng, as it also contains a high ginsenoside content and exhibits similar biological activities.     

Simultaneous measurement of three-dimensional joint kinematics and ligament strains with optical methods

The objective of this study was to assess the precision and accuracy of a nonproprietary, optical three-dimensional (3D) motion analysis system for the simultaneous measurement of soft tissue strains and joint kinematics. The system consisted of two high-resolution digital cameras and software for calculating the 3D coordinates of contrast markers. System precision was assessed by examining the variation in the coordinates of static markers over time. Three-dimensional strain measurement accuracy was assessed by moving contrast markers fixed distances in the field of view and calculating the error in predicted strain. Three-dimensional accuracy for kinematic measurements was assessed by simulating the measurements that are required for recording knee kinematics. The field of view (190 mm) was chosen to allow simultaneous recording of markers for soft tissue strain measurement and knee joint kinematics. Average system precision was between +/-0.004 mm and +/-0.035 mm, depending on marker size and camera angle. Absolute error in strain measurement varied from a minimum of +/-0.025% to a maximum of +/-0.142%, depending on the angle between cameras and the direction of strain with respect to the camera axes. Kinematic accuracy for translations was between +/-0.008 mm and +/-0.034 mm, while rotational accuracy was +/-0.082 deg to +/-0.160 deg. These results demonstrate that simultaneous optical measurement of 3D soft tissue strain and 3D joint kinematics can be performed while achieving excellent accuracy for both sets of measurements.