Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells

We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.     

Genetic footprints of late Quaternary climate change in the diversity of Patagonian‐Fueguian rodents

Species are impacted by climate change at both ecological and evolutionary time scales. Studies in northern continents have provided abundant evidence of dramatic shifts in distributions of species subsequent to the last glacial maximum (LGM), particularly at high latitudes. However, little is known about the history of southern continents, especially at high latitudes. South America is the only continent, other than Antarctica, that extends beyond 40 °S. Genetic studies of a few Patagonian species have provided seemingly conflicting results, indicating either postglacial colonization from restricted glacial refugia or persistence through glacial cycles and in situ differentiation. Using mitochondrial DNA sequences of 14 species of sigmodontine rodents, a major faunal ensemble of Patagonia and Tierra del Fuego, we show that at least nine of these species bear genetic footprints of demographic expansion from single restricted sources. However, timing of demographic expansion precedes the LGM in most of these species. Four species are fragmented phylogeographically within the region. Our results indicate that (i) demographic instability in response to historical climate change has been widespread in the Patagonian‐Fueguian region, and is generally more pronounced at high latitudes in both southern and northern continents; (ii) colonization from lower latitudes is an important component of current Patagonian‐Fueguian diversity; but (iii) in situ differentiation has also contributed to species diversity.     

Measuring and modelling the transport and root uptake of chemicals in the unsaturated zone

To determine the mechanisms prescribing the movement and uptake of chemicals in the soil of the rootzone, controlled experiments were carried out in four lysimeters growing tomatoes. Each lysimeter had a depth-wise array of 9 Time Domain Reflectometry (TDR) probes to monitor the soil's water content. Chloride was used as an inert tracer, and was applied with the nutrient solution used for irrigation. Sulphate was used as a reactive tracer, and was applied as a pulse resident in the upper 100 mm of the soil. The measured water contents and the concentrations of the chemicals in the soil profile at the end of the experiment were compared to a deterministic model based on Richards' equation and the convection–dispersion equation linked with various macroscopic sink terms for root water and chemical uptake. The uptake function based on matric pressure head seems to describe the uptake of water and chemicals of our tomato plants best. At high soil solution concentration chloride and sulphate exclusion occurred. Our simple model could be used to describe the major features of coupled water and chemical uptake. However, our approach of inverse modelling to infer the parameters for solute transport and root uptake could not be used to distinguish between soil-based mechanisms and plant uptake mechanisms. The choice of the root water uptake model had only a small effect on the final water content profiles, but led to differences in the final solute profiles of sulphur and chloride. This indicates that tracers might provide improved determination of the uptake mechanisms.     

Intracellular phosphorylation of benzyladenosine is related to apoptosis induction in tobacco BY-2 cells

Treatment of tobacco BY‐2 cells with micromolar concentration of benzyladenosine ([9R]BA) resulted in the loss of cell viability in a time‐ and concentration‐dependent manner. Cell death induced by [9R]BA exhibited typical apoptotic hallmarks including cell shrinkage, chromatin condensation and degradation of nuclear DNA to characteristic high molecular weight (HMW) as well as nucleosomal size fragments. Externally added [9R]BA was very rapidly and almost quantitatively phosphorylated within BY‐2 cells. Accumulation of [9R]BA‐monophosphate was accompanied by massive production of endogenous reactive oxygen species (ROS), intracellular ATP depletion, and these events were followed by the loss of cell viability. Inhibition of intracellular phosphorylation of [9R]BA by adenosin kinase inhibitor, 5′‐amino‐5′‐deoxyadenosine (AdAs), diminished ROS production, ATP depletion, and consequently prevented cells from death. Selective inhibition of ROS production without restoring ATP production, however, did not provide any protection to cells. In contrast, even enhanced phosphorylation of [9R]BA caused by adenosine that simultaneously revived ATP synthesis reduced the number of dying cells. This is the first evidence of a direct relationship between intracellular phosphorylation of [9R]BA and apoptosis induction in BY‐2 cells. ATP depletion but not ROS production is the key secondary event that determines the cellular decision between life and death.     

A cyanobacterial bloom prevents fish trophic cascades

1. We experimentally compared the impacts of visually feeding zooplanktivorous fish and filter‐feeding omnivorous fish in shallow tropical Dakar Bango reservoir, Senegal. We provoked a cyanobacterial Anabaena bloom under mesotrophic to eutrophic N‐limited conditions in 18 enclosures assigned to six Nile tilapia life‐stage treatments, at typical biomasses: fishless control (C), zooplanktivorous fry (Z), omnivorous juveniles (O), herbivorous fingerlings (H) and two combinations (OZ, OH). 2. All fish grew well, but as prevalent inedible phytoplankton dampened fish effects, community‐level trophic cascades did not occur. Planktivore types acted independently and affected differentially the biomasses of total zooplankton, cyclopoids, nauplii, cladocerans, invertebrate carnivores, large herbivores, colonial cyanobacteria and Chlorophyta. They neither influenced the total biomass of phytoplankton, nor most water chemistry characteristics. Responses were apparently not fish‐biomass related. The bloom collapsed synchronously in all enclosures, coinciding with enrichment ending, with a return to clear water within 12 days. 3. Our results support the hypothesis that excess nutrients and prevalent inedible cyanobacteria inhibit the cascading effects of natural biomass levels of both visually feeding zooplanktivores and filter‐feeding omnivores. In N‐limited meso‐eutrophic shallow tropical lakes with predominantly small herbivorous zooplankton, neither the type nor the biomass of planktivorous fish present seems likely to prevent the transient outburst of cyanobacterial blooms. Such fragile ecosystems may thus not sustain a trophic state suitable for drinking water production, unless human impacts are restricted. The generality of restoration approaches based on ecological engineering should be further explored.     

Plant uptake and leaching of copper during EDTA-enhanced phytoremediation of repacked and undisturbed soil

EDTA-enhanced phytoremediation of copper contaminated soil was evaluated. Up to 740 g g^–1 of Na₂H₂ EDTA in solution was added to repacked soil columns, and intact cores of a sandy loam of volcanic origin, that was growing Agrostis tenuis. The soil contained up to 400 g g^–1 of copper due to a history of fungicide spraying. EDTA application increased the herbage copper concentration of the grass growing in repacked soil from 30 to 300 g g^–1, but the same application to an intact core only brought about an increase from 10 to 60 g g^–1. More copper accumulated in the herbage when the EDTA was applied in numerous small doses than in just one or two larger amounts. Calculation of the concentration of copper in the water taken up by the grass revealed this to be two orders of magnitude lower than that in the soil solution. As a result of the EDTA applications, about 100 times more copper was leached than was taken up by the herbage. This means that a strategy for managing leaching losses needs to be part of any plan for EDTA-enhanced phytoremediation.     

Open Access to essential health care information

Open Access publishing is a valuable resource for the synthesis and distribution of essential health care information. This article discusses the potential benefits of Open Access, specifically in terms of Low and Middle Income (LAMI) countries in which there is currently a lack of informed health care providers - mainly a consequence of poor availability to information. We propose that without copyright restrictions, Open Access facilitates distribution of the most relevant research and health care information. Furthermore, we suggest that the technology and infrastructure that has been put in place for Open Access could be used to publish download-able manuals, guides or basic handbooks created by healthcare providers in LAMI countries.     

Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium

A new selective agar medium, ALOA, for the selective and differential isolation of Listeria monocytogenes has been evaluated. All stressed cultures of L. monocytogenes serovars tested grew on the medium as bluish colonies surrounded by a distinctive opaque halo and gave a productivity ratio of at least 0.95. Non-pathogenic Listeria sp. produced bluish colonies without a halo as was also the case for some enterococci and bacilli. Special attention must be paid to some Bacillus cereus strains and L. ivanovii since their colony appearance can be misleading. Only some unidentified listeria-like bacteria gave false-positive results. ALOA detected 4. 3% more positives from naturally contaminated dairy and meat samples compared with the ISO procedure when used with GenprobeTM or VidasTM for confirmation of presumptive colonies; 13.9% false negatives were found compared with 38.9% using PALCAM/Oxford. ALOA was also clearly superior to Oxford and PALCAM when samples containing both L. monocytogenes and L. innocua were examined. The introduction of ALOA in standard isolation procedures as an additional medium would enhance the detection ratio and reduce the time and cost of analysis for L. monocytogenes.     

ZUR AUSLÖSUNG DER EIABLAGE BEI DER APHIDOPHAGEN GALLMÜCKE APHIDOLETES APHIDIMYZA (DIPTERA: CECIDOMYIIDAE)

Die Eiablage von Aphidoletes aphidimyza (Rond.) konnte im Experiment durch Honigtau oder durch tote Blattläuse (Myzus persicae, Aphis fabae und Acyrthosiphon pisum), jedoch wesentlich stärker durch beide zusammen ausgelöst werden. Siphonensekret von Acyrthosiphon pisum wurde ebenfalls belegt. Dagegen gelang es nicht, die Gallmücken durch klebrige Substanzen, deren Konsistenz der des Honigtaus ähnlich ist, zur Eiablage zu bringen. Während Exuvien der Erbsenblattlaus für sich allein fast keine Wirkung zeigten, lösten sie in Kombination mit Honigtau die Eiablage ebenso stark aus wie die toten Blattläuse mit Honigtau. Dagegen waren kleine Steinchen in Blattlaus-Größe anstelle der Exuvien unwirksam. Auch Duftstoffe von Blattläusen und Honigtau führten nicht zur Eiablage. Von den Bestandteilen des Honigtaus waren bestimmte Kohlehydrate und Aminosäuren schwach, andere, vor allem Fruktose und Arginin, besser wirksam; reproduzierbar war die Wirkung nur in Kombination mit getöteten Blattläusen.