Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae

The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H(+) symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H(+)/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.     

A comparison of the strength of biodiversity effects across multiple functions

In order to predict which ecosystem functions are most at risk from biodiversity loss, meta-analyses have generalised results from biodiversity experiments over different sites and ecosystem types. In contrast, comparing the strength of biodiversity effects across a large number of ecosystem processes measured in a single experiment permits more direct comparisons. Here, we present an analysis of 418 separate measures of 38 ecosystem processes. Overall, 45 % of processes were significantly affected by plant species richness, suggesting that, while diversity affects a large number of processes not all respond to biodiversity. We therefore compared the strength of plant diversity effects between different categories of ecosystem processes, grouping processes according to the year of measurement, their biogeochemical cycle, trophic level and compartment (above- or belowground) and according to whether they were measures of biodiversity or other ecosystem processes, biotic or abiotic and static or dynamic. Overall, and for several individual processes, we found that biodiversity effects became stronger over time. Measures of the carbon cycle were also affected more strongly by plant species richness than were the measures associated with the nitrogen cycle. Further, we found greater plant species richness effects on measures of biodiversity than on other processes. The differential effects of plant diversity on the various types of ecosystem processes indicate that future research and political effort should shift from a general debate about whether biodiversity loss impairs ecosystem functions to focussing on the specific functions of interest and ways to preserve them individually or in combination.     

Affective response to a loved one's pain: insula activity as a function of individual differences

Individual variability in emotion processing may be associated with genetic variation as well as with psychological predispositions such as dispositional affect styles. Our previous fMRI study demonstrated that amygdala reactivity was independently predicted by affective-cognitive styles (phobic prone or eating disorders prone) and genotype of the serotonin transporter in a discrimination task of fearful facial expressions. Since the insula is associated with the subjective evaluation of bodily states and is involved in human feelings, we explored whether its activity could also vary in function of individual differences. In the present fMRI study, the association between dispositional affects and insula reactivity has been examined in two groups of healthy participants categorized according to affective-cognitive styles (phobic prone or eating disorders prone). Images of the faces of partners and strangers, in both painful and neutral situations, were used as visual stimuli. Interaction analyses indicate significantly different activations in the two groups in reaction to a loved one's pain: the phobic prone group exhibited greater activation in the left posterior insula. These results demonstrate that affective-cognitive style is associated with insula activity in pain empathy processing, suggesting a greater involvement of the insula in feelings for a certain cohort of people. In the mapping of individual differences, these results shed new light on variability in neural networks of emotion.     

Chemistry of Two Distinct Aeolid Spurilla Species: Ecological Implications

The lipophilic extracts of two marine aeolid nudibranch molluscs of the genus Spurilla collected in distinct geographical areas have been chemically analyzed. The Et₂O extracts of the nudibranchs were dominated by the presence of usual fatty acids and sterols and contained terpenoid compounds 1  –  3 as minor metabolites. Spurillin A ( 1 ) and spurillin B ( 3 ) were new molecules whereas cis‐γ‐monocyclofarnesol ( 2 ) was already reported in the literature as a synthesis product. Interestingly, bursatellin ( 4 ), previously isolated from anaspidean molluscs of the genus Bursatella, was found in the butanol extract of both Spurilla species. Compounds 1  –  4 were not detected in the extracts of the sea‐anemone preys collected together with the molluscs.     

Structural and Functional Alterations of Subcutaneous Small Resistance Arteries in Severe Human Obesity

Obese persons are at increased cardiovascular risk and exhibit increased arterial stiffness and impaired endothelial function of large‐ and medium‐size arteries. We hypothesized that normotensive subjects suffering from severe obesity would also present remodeling and endothelial dysfunction of small resistance arteries. A total of 16 lean (age: 49.6 ± 2.9 years, BMI: 22.9 ± 0.3 kg/m², mean ± s.e.m.) and 17 age‐matched severely obese (BMI: 41.1 ± 2.3 kg/m²) normotensive subjects were investigated. None had glucose or lipid metabolic abnormalities except for insulin resistance. Resistance arteries, dissected from abdominal subcutaneous tissue, were assessed on a pressurized myograph. For superimposable blood pressure, the media thickness, media cross‐sectional area (CSA), and media‐to‐lumen ratio values of resistance arteries were markedly and significantly greater in obese compared to lean subjects (media thickness 26.3 ± 0.6 vs. 16.2 ± 0.6 µm, CSA 22,272 ± 1,339 vs. 15,183 ± 1,186 µm², and media‐to‐lumen ratio 0.113 ± 0.006 vs. 0.059 ± 0.001, respectively, P < 0.01). Acetylcholine‐induced relaxation was impaired in vessels from obese subjects compared to the lean individuals (?40.4 ± 1.3%, P < 0.01), whereas endothelium‐independent vasorelaxation was similar in all groups. Stiffness of small arteries as assessed by the stress/strain relationship was similar in lean and severely obese subjects. We conclude that severe human obesity is associated with profound alterations in structural and functional characteristics of small arteries, which may be responsible for the presence of elevated cardiovascular risk and increased incidence of coronary, cerebrovascular and renal events reported in obesity.     

Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment

Background

Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4⁺, murine CD4^−/−, HLA-DR3⁺ mice.

Methodology/Principal Findings

Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45⁺ showed depletion of CD4⁺, CD8⁺, CD19⁺, and CD117⁺ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4⁺ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death.

Conclusions/Significance

NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.     

Using Satellite Tracking and Isotopic Information to Characterize the Impact of South American Sea Lions on Salmonid Aquaculture in Southern Chile

Apex marine predators alter their foraging behavior in response to spatial and/or seasonal changes in natural prey distribution and abundance. However, few studies have identified the impacts of aquaculture that represents a spatially and temporally predictable and abundant resource on their foraging behavior. Using satellite telemetry and stable isotope analysis we examined the degree of spatial overlap between the South American sea lion (SASL) and salmon farms, and quantify the amount of native prey versus farmed salmonids in SASL diets. We instrumented eight SASL individuals with SRDL-GPS tags. Vibrissae, hair and skin samples were collected for δ¹³C and δ¹⁵N analyses from five of the tagged individuals and from four males captured in a haul-out located adjacent to salmon farms. Tracking results showed that almost all the foraging areas of SASL are within close proximity to salmon farms. The most important prey for the individuals analyzed was farmed salmonids, with an estimated median (±SD) contribution of 19.7 ± 13.5‰ and 15.3 ± 9.6‰ for hair and skin, respectively. Using vibrissae as a temporal record of diet for each individual, we observed a remarkable switch in diet composition in two SASL, from farmed salmonids to pelagic fishes, which coincided with the decrease of salmon production due to the infectious salmon anemia virus that affected salmon farms in Chile at the end of 2008. Our study demonstrates the usefulness of integrating stable isotope derived dietary data with movement patterns to characterize the impacts of a non-native prey on the foraging ecology of an apex marine predator, providing important applied implications in situations where interactions between aquaculture and wildlife are common.     

Circular dichroism study of ribonuclease A mutants containing the minimal structural requirements for dimerization and swapping

Four residues Pro19, Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins, studied by means of CD measurements, proved reversible and well represented by the two-state ND transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase.     

Identifying protein construct variants with increased crystallization propensity--a case study

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.