Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein

Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in resurrection plants and in plant seeds, and the recent discovery of a dehydration-induced Group 3 LEA-like gene in the nematode Aphelenchus avenae suggests a similar association in anhydrobiotic animals. Despite their importance, little is known about the structure of Group 3 LEA proteins, although computer modeling and secondary structure algorithms predict a largely alpha-helical monomer that forms coiled coil oligomers. We have therefore investigated the structure of the nematode protein, AavLEA1, in the first such analysis of a well characterized Group 3 LEA-like protein. Immunoblotting and subunit cross-linking experiments demonstrate limited oligomerization of AavLEA1, but analytical ultracentrifugation and gel filtration show that the vast majority of the protein is monomeric. Moreover, CD, fluorescence emission, and Fourier transform-infrared spectroscopy indicate an unstructured conformation for the nematode protein. Therefore, in solution, no evidence was found to support structure predictions; instead, AavLEA1 seems to be natively unfolded with a high degree of hydration and low compactness. Such proteins can, however, be induced to fold into more rigid structures by partner molecules or by altered physiological conditions. Because AavLEA1 is associated with desiccation stress, its Fourier transform-infrared spectrum in the dehydrated state was examined. A dramatic but reversible increase in alpha-helix and, possibly, coiled coil formation was observed on drying, indicating that computer predictions of secondary structure may be correct for the solid state. This unusual finding offers the possibility that structural shifts in Group 3 LEA proteins occur on dehydration, perhaps consistent with their role in anhydrobiosis.     

Cofactors required for TLR7- and TLR9-dependent innate immune responses

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Highlights? RNAi and systems-based analysis identified 190 TLR7/9 signaling cofactors ? Pathway crossprofiling enabled mapping cofactors to specific NFκB signaling modules ? HRS is a mediator of ubiquitin-dependent nondegradative endosomal TLR sorting     

The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch

Tryptophan (Trp) is abundant in membrane proteins, preferentially residing near the lipid–water interface where it is thought to play a significant anchoring role. Using a total of 3 μs of molecular dynamics simulations for a library of hydrophobic WALP-like peptides, a long poly-Leu α-helix, and the methyl-indole analog, we explore the thermodynamics of the Trp movement in membranes that governs the stability and orientation of transmembrane protein segments. We examine the dominant hydrogen-bonding interactions between the Trp and lipid carbonyl and phosphate moieties, cation–π interactions to lipid choline moieties, and elucidate the contributions to the thermodynamics that serve to localize the Trp, by ~ 4 kcal/mol, near the membrane glycerol backbone region. We show a striking similarity between the free energy to move an isolated Trp side chain to that found from a wide range of WALP peptides, suggesting that the location of this side chain is nearly independent of the host transmembrane segment. Our calculations provide quantitative measures that explain Trp's role as a modulator of responses to hydrophobic mismatch, providing a deeper understanding of how lipid composition may control a range of membrane active peptides and proteins.     

The chemistry of uranium dispersion in groundwaters at the Pinhal do Souto Mine,Portugal

Solution studies of H-autunite, H₂(UO₂)₂(PO₄)₂, and its K⁺, Na⁺, Li⁺, Ca²⁺, Mg²⁺, Cu²⁺ and Ni²⁺ analogues, have been carried out and solution stability constants determined. These values are in excellent agreement with those recently reported by other workers at 298.2 K and ionic strength equal to zero. separate field study involving groundwaters collected from the oxidized zone of the Pinhal do Souto mine, Portugal, has been undertaken. Here the principal uranium minerals are autunite, Ca(UO₂)₂(PO₄)₂·nH₂O and torbernite Cu(UO₂)₂(PO₄)₂·nH₂O. Detailed analyses of and calculations of species distributions in these samples indicate that currently circulating groundwaters are undersaturated with respect to these two minerals by two to four orders of magnitude. The mineralogical and geochemical significance of these results is discussed.     

Functional groups of woody species in semi‐arid regions at low latitudes

In seasonally dry environments, woody species have different survival strategies. However, little is known about how environmental variables affect the phenology and water dynamics of these species. We aim to understand which variables initiate the vegetative phenophases of species in a tropical semiarid climate at 3°S latitude, where variation in photoperiod is minimal and rainfall is seasonal. We hypothesize that groups of species with similar vegetative phenologies, under similar conditions, are functionally similar in terms of water storage and use. We analyse the relationship between functional characteristics related to the acquisition and utilization of water, such as wood density, water storage capacity, water potential and vegetative phenology. The attributes were ordered by multidimensional scaling, and a multiple response permutation procedure was used to test consistency of the groups. Canonical correspondence analysis and Mantel tests were used to evaluate the phenophase response to environmental variables. We found four functional groups: (i) deciduous low wood density, which lose 75% of their leaves one month before the end of the rains; (ii) evergreen high wood density; (iii) early deciduous high wood density, which lose 75% of their leaves one month after the end of the rains; and (iv) late deciduous high wood density, which lose 75% of their leaves two months after the end of the rains. As expected, the vegetative phenodynamics of the deciduous high wood density group were mainly influenced by water availability. The evergreens did not show a correlation with rainfall. Only leaf shedding of the late deciduous, and the vegetative phenophases of the evergreens, responded to an increase in temperature and photoperiod. Bud‐break responded to increased photoperiod and soil humidity in the deciduous low wood density group. The foliar periodicity groups can be explained by the presence of species that differ mainly in their mechanisms of water acquisition and use.     

Quantification of meniscal volume by segmentation of 3T magnetic resonance images

Meniscal injuries place the knee at risk for early osteoarthritis (OA) because they disrupt their load-bearing capabilities. Partial resection is routinely performed to alleviate symptomatic meniscal tears. While the removal of meniscal tissue may not be the only factor associated with partial meniscectomy outcome, the amount removed certainly contributes to functional loss. It is unknown, however, whether there is a critical amount of meniscal tissue that can be removed without diminishing the structure's chondroprotective role. In order to examine the existence of such a threshold, it is necessary to accurately quantify meniscal volume both before and after partial meniscectomy to determine the amount of meniscal tissue removed. Therefore, our goal was to develop and validate an MR-based method for assessing meniscal volume. The specific aims were: (1) to evaluate the feasibility of the MR-based segmentation method; (2) to determine the method's reliability for repeated measurements; and (3) to validate its accuracy in situ. MR images were obtained on a 3T magnet, and each scan was segmented using a biplanar approach. The MR-based volumes for each specimen were compared to those measured by water displacement. The results indicate that the biplanar approach of measuring meniscal volumes is accurate and reliable. The calculated volumes of the menisci were within 5% of the true values, the coefficients of variation were 4%, and the intraclass correlation coefficients were greater than 0.96. These data demonstrate that this method could be used to measure the amount of meniscal tissue excised during partial meniscectomy to within 125.7 mm(3).     

Cell therapy with bone marrow stromal cells after intracerebral hemorrhage: impact of platelet-rich plasma scaffolds

Background aimsCell therapy using bone marrow stromal cells (BMSCs) has been considered a promising strategy for neurologic sequelae after intracerebral hemorrhage (ICH). However, after intracerebral administration of BMSCs, most of the cells die, partly because of the absence of extracellular matrix. Intracerebral transplantation of BMSCs, supported in a platelet-rich plasma (PRP) scaffold, optimizes this type of cell therapy.MethodsICH was induced by stereotactic injection of 0.5 IU of collagenase type IV in the striatum of adult Wistar rats (n = 40). Two months later, the rats were subjected to intracerebral administration of 5 × 10⁶ allogeneic BMSCs embedded in a PRP scaffold (n = 10), 5 × 10⁶ allogeneic BMSCs in saline (n = 10), PRP-derived scaffold only (n = 10) or saline only (n = 10). Functional improvements in each group over the next 6 months were assessed using Rotarod and Video-Tracking-Box tests. Endogenous neurogenesis and survival of transplanted BMSCs were examined at the end of follow-up.ResultsOur study demonstrated neurologic improvement after BMSC transplantation and significantly better functional improvement for the group of animals that received BMSCs in the PRP-derived scaffold compared with the group that received BMSCs in saline. Histologic results showed that better functional outcome was associated with strong activation of endogenous neurogenesis. After intracerebral administration of BMSCs, donor cells were integrated in the injured tissue and showed phenotypic expression of glial fibrillary acidic protein and neuronal nucleus.ConclusionsPRP-derived scaffolds increase the viability and biologic activity of BMSCs and optimize functional recovery when this type of cell therapy is applied after ICH.     

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.     

Shrub emergence and seedling growth in microenvironments created by Prosopis glandulosa

Abstract. Facilitation of the establishment of certain plant species by nurse plants is a common phenomenon in arid and semiarid ecosystems. The most commonly reported mechanisms of facilitation include cooler temperatures and increased soil nutrients beneath the nurse plant canopy, which favor establishment of other plant species. During conversion of upland grasslands to thorn woodland in southern Texas, Prosopis glandulosa appears to facilitate establishment of other woody plants, including Celtis pallida, whereas Acacia smallii occurs only in habitats between P. glandulosa canopies. We tested the hypothesis that light intensity and soils under P. glandulosa canopies facilitate seedling emergence and growth of C. pallida but inhibit seedling emergence and growth of A. smallii. In the field, C. pallida and A. smallii seeds were planted under P. glandulosa canopies and in adjacent interspaces. Percent emergence of C. pallida seedlings was greater under the canopy of P. glandulosa, whereas percent emergence of A. smallii seedlings was greater in interspaces. In a greenhouse experiment, seeds of each species were planted in pots filled with soil from under P. glandulosa canopies or from adjacent interspaces. Two treatments, shade and sunlight, were imposed and plants harvested seven weeks later. Seedling mass of both species was greater in canopy soil than in interspace soil in sunlight but mass of the two species did not differ between soil sources in shade. Canopy soils contained more total and available nitrogen than interspace soils. These results suggest that light is more limiting than nutrients under shaded conditions and so neither species can take advantage of the high nutrients beneath P. glandulosa. Shade and greater soil nutrients beneath P. glandulosa do not appear to be the major factors that facilitate C. pallida or inhibit A. smallii. Aggregation of C. pallida beneath P. glandulosa canopies appears to be a complex process that involves both passive facilitation (seed dispersal by birds) and active facilitation (reduction of seed dormancy by under-canopy temperatures) operating only during the seed germination stage with successional mechanisms other than facilitation operating during later stages of shrub establishment and growth.     

Radiobrominated m-tyrosine analog as potential CNS L-dopa pet tracer

Radiobrominated 6-bromo-m-tyrosine (6-BMT) was prepared and the time course of its localization in selected cerebral and peripheral organs in the mouse was determined. Since m-tyrosine is known to have L-dopa-like properties in vivo, our goal was to assess the utility of a radiolabeled analog as a tracer for cerebral L-dopa. Our preliminary results showed that substantial amounts of 6-BMT is extracted by the mouse brain and that the regional distribution and time course of the radiotracer is consistent with uptake in regions rich in dopamine neurons. Although a more thorough biochemical characterization of 6-BMT is necessary, this or other positron emitting analogs of m-tyrosine, such as an 18F labelled analog, may be useful PET tracers for the non-invasive study of dopamine turnover in humans.