Raman optical activity and circular dichroism reveal dramatic differences in the influence of divalent copper and manganese ions on prion protein folding

The binding of divalent copper ions to the full-length recombinant murine prion protein PrP23-231 at neutral pH was studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UV CD). The effect of the Cu2+ ions on PrP structure depends on whether they are added after refolding of the protein in water or are present during the refolding process. In the first case ROA reveals that the hydrated alpha-helix is lost, with UV CD revealing a drop from approximately 25% to approximately 18% in the total alpha-helix content. The lost alpha-helix could be that comprising residues 145-156, located within the region associated with scrapie PrP formation. In the second case, ROA reveals the protein's structure to be almost completely disordered/irregular, with UV CD revealing a drop in total alpha-helix content to approximately 5%. Hence, although Cu2+ binding takes place exclusively within the unfolded/disordered N-terminal region, it can profoundly affect the structure of the folded/alpha-helical C-terminal region. This is supported by the finding that refolding in the presence of Cu2+ of a mutant in which the first six histidines associated with copper binding to the N-terminal region are replaced by alanine has a similar alpha-helix content to the metal-free protein. In contrast, when the protein is refolded in the presence of divalent manganese ions, ROA indicates the alpha-helix is reinforced, with UV CD revealing an increase in total alpha-helix content to approximately 30%. The very different influence of Cu2+ and Mn2+ ions on prion protein structure may originate in the different stability constants and geometries of their complexes.     

A multiple watershed field test of hydrogeomorphic functional assessment of headwater streams—Variability in field measurements between independent teams

Ephemeral and intermittent headwater streams are under increasing pressure from disturbance and development. Rapid, repeatable assessment techniques are needed in order to gauge the condition of these stream systems. Several attributes of these headwater streams constrain the use of the most widely used macroinvertebrate or water quality stream assessment techniques. The hydrogeomorphic (HGM) functional assessment is a reference-based alternative technique. To evaluate this alternative, repeated assessments were conducted in eight high-gradient headwaters in West Virginia by four independent teams. Across-site and measurement variance among teams was assessed using a coefficient of variation (CV, expressed as percent). A variability of >50% CV, which suggests less repeatable results, occurred in only 13.8% of measurements, primarily associated with 2 of the 9 variables examined (snag density and substrate size). Between site measurement variance was the greatest at more highly disturbed sample locations, particularly with regard to the large woody debris, tree species richness, and channel bank erosion variables. Variables with the lowest CV were tree diameter, detrital cover, canopy cover, and channel embeddedness. Based on these results, measurements included when applying HGM approach to these streams should focus on direct measurements or directed estimates that yield a large response range across a spectrum of sites while maintaining consistent repeatability among different teams, with special attention paid to their use in highly disturbed sites.     

Evaluation of the predatory mite Amblyseius hainanensis (Acari: Phytoseiidae) and artificial rainfall for the management of Brevipalpus obovatus (Acari: Tenuipalpidae)

Brevipalpus obovatus Donnadieu is an important pest mite on tea plants in South China. In the current study, predatory mites of B. obovatus in the tea gardens of Guangzhou were extensively surveyed. In total, 13 species of predatory mites (four families with seven genera) were recorded. The population proportion of Amblyseius hainanensis Wu et Qian was the highest (68.6?%), followed by that of Anystis baccarum (L.) (8.4?%) and A. theae Wu (6.3?%). The effects of starvation time, habitat size and pest population density on the predatory efficiency of the most dominant species, A. hainanensis, feeding on B. obovatus were assessed. In addition, the effectiveness of artificial rainfall in reducing B. obovatus populations was evaluated. After starvation for 48?h, the predatory efficiency of A. hainanensis was significantly higher than those that had been starved for 24 or 72?h when 30-50 B. obovatus eggs were made available. The predation of A. hainanensis on B. obovatus also increased with increasing prey density. The number of prey attacked by A. hainanensis in a 3.2?cm(2) habitat was significantly higher than in a 6.3?cm(2) habitat. The average predation of A. hainanensis was 31.7 eggs per day when offered 100 B. obovatus eggs on a tea leaf. This decreased to 17.8 eggs per day when four A. hainanensis shared 100 B. obovatus eggs. B. obovatus populations can be reduced by artificial rainfall, with the reduction affected by rainfall intensity. With an intensity of 40?mm in 15?min, 90.2?% mortality of B. obovatus occurred; lower mortalities were recorded (13.3 and 29.8?%) when the intensity was 2 or 4?mm in 15?min. Combination of the predatory mite A. hainanensis and artificial rainfall for the integrated pest management of B. obovatus is discussed.     

Importance of organosulfur utilization for survival of Pseudomonas putida in soil and rhizosphere

The sulfur present in both agricultural and uncultivated soils is largely in the form of sulfonates and sulfate esters and not as free, bioavailable inorganic sulfate. Desulfurization of the former compounds in vitro has previously been studied in Pseudomonas putida, a common rhizosphere inhabitant. Survival of P. putida strains was now investigated in three sulfur-deficient Danish soils which were found to contain 60 to 70% of their sulfur in sulfonate or sulfate ester form, as determined by X-ray near-edge spectroscopy. The soil fitness of P. putida S-313 was compared with that of isogenic strains with mutations in the sftR and asfA genes (required for in vitro desulfurization of sulfate esters and arylsulfonates, respectively) and in the ssu locus (required in vitro for the desulfurization of both sulfonates and sulfate esters). asfA or sftR mutants showed significantly reduced survival compared to the parent strain in bulk soil that had been enriched with carbon and nitrogen to mimic rhizosphere conditions, but this reduced survival was not observed in the absence of these additives. In a tomato rhizosphere grown in compost, survival of sftR and ssu mutants was reduced relative to the parent strain. The results demonstrate that the ability to desulfurize sulfonates and sulfate esters is critical for survival of bacteria in the rhizosphere but less so in bulk soils outside the influence of plant roots, where carbon is the limiting nutrient for growth.     

An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons

Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll^® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.     

Transmission Blocking Immunity in the Malaria Non-Vector Mosquito Anopheles quadriannulatus Species A

Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa, Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vector mosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus is largely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodium berghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits and manifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequent stages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and the thioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completely abolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatus into a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of natural refractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to control transmission of the disease.     

Patterns and controls of the variability of radiation use efficiency and primary productivity across terrestrial ecosystems

Aim The controls of gross radiation use efficiency (RUE), the ratio between gross primary productivity (GPP) and the radiation intercepted by terrestrial vegetation, and its spatial and temporal variation are not yet fully understood. Our objectives were to analyse and synthesize the spatial variability of GPP and the spatial and temporal variability of RUE and its climatic controls for a wide range of vegetation types. Location A global range of sites from tundra to rain forest. Methods We analysed a global dataset on photosynthetic uptake and climatic variables from 35 eddy covariance (EC) flux sites spanning between 100 and 2200 mm mean annual rainfall and between ?13 and 26°C mean annual temperature. RUE was calculated from the data provided by EC flux sites and remote sensing (MODIS). Results Rainfall and actual evapotranspiration (AET) positively influenced the spatial variation of annual GPP, whereas temperature only influenced the GPP of forests. Annual and maximum RUE were also positively controlled primarily by annual rainfall. The main control parameters of the growth season variation of gross RUE varied for each ecosystem type. Overall, the ratio between actual and potential evapotranspiration and a surrogate for the energy balance explained a greater proportion of the seasonal variation of RUE than the vapour pressure deficit (VPD), AET and precipitation. Temperature was important for determining the intra‐annual variability of the RUE at the coldest energy‐limited sites. Main conclusions Our analysis supports the idea that the annual functioning of vegetation that is adapted to its local environment is more constrained by water availability than by temperature. The spatial variability of annual and maximum RUE can be largely explained by annual precipitation, more than by vegetation type. The intra‐annual variation of RUE was mainly linked to the energy balance and water availability along the climatic gradient. Furthermore, we showed that intra‐annual variation of gross RUE is only weakly influenced by VPD and temperature, contrary to what is frequently assumed. Our results provide a better understanding of the spatial and temporal controls of the RUE and thus could lead to a better estimation of ecosystem carbon fixation and better modelling.