A New Species of Goniopholis from the Upper Jurassic of Portugal

The new goniopholidid crocodile Goniopholis baryglyphaeus sp. nov. from the Kimmeridgian of Guimarota/Leiria, Portugal is based on the oldest known relatively complete skeleton of Goniopholis from Europe and consists of a nearly complete skull together with a mandible and various postcranial remains. G. baryglyphaeus shows characters that hitherto have only been described for Goniopholis simus : a caudal expansion of the prefrontal that excludes the frontal from the medial margin of the orbit; and the loss of contact between the paroccipital process and the lateral surface of the squamosal and the quadrate, which exposes the cranioquadrate canal laterally. This new species can be distinguished from other European Goniopholis species by an articular facet placed ventral to the retroarticular process, and directed slightly medioventrally; a caudal margin of the mandible forming almost a right angle at its caudoventral corner; an almost square rostrolateral corner of the cranial table; a straight transverse suture between the parietal and the frontal with a median rostral process; a rostrally tapering wedge-like lacrimal, and a regular heavy skull-sculpturing. The new find of Goniopholis in Portugal shows that this genus was distributed in a larger area, and was present much earlier, than hitherto reported in south-western Europe.     

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.     

The osmolyte taurine protects against ultraviolet B radiation-induced immunosuppression

Organic osmolytes, such as taurine, are involved in cell volume homeostasis and cell protection. Epidermal keratinocytes possess an osmolyte strategy, i.e., they take up taurine upon hyperosmotic stress and express the corresponding transporter TAUT. UVB irradiation also triggers taurine uptake and TAUT expression in this cell type. We therefore asked whether taurine plays a role in photoprotection. By using a TAUT-deficient mouse model, lack of taurine in the skin was found to cause a significantly higher sensitivity to UVB-induced immunosuppression. This was not due to an increased generation or decreased repair of UVB-induced DNA photoproducts in the skin of these animals. Instead, decreased skin taurine levels were associated with an increased formation of the soluble immunosuppressive molecule platelet-activating factor (PAF) from the membranes of UVB-irradiated epidermal cells. Blocking PAF activity in taut-deficient mice with a PAF receptor antagonist abrogated their increased sensitivity to UVB-induced immunosuppression. Moreover, taut -/- mice were more sensitive to PAF-mediated immunosuppression than taut +/+ mice. These data suggest that taurine uptake by epidermal cells prevents undue PAF formation, and thereby photoimmunosuppression. Thus, similar to nucleotide excision repair, taurine uptake is critically involved in photoprotection of the skin.     

A method for extracting plant roots from soil which facilitates rapid sample processing without compromising measurement accuracy

This study evaluates a novel method for extracting roots from soil samples and applies it to estimate standing crop root mass (+/- confidence intervals) in an eastern Amazon rainforest. Roots were manually extracted from soil cores over a period of 40 min, which was split into 10 min time intervals. The pattern of cumulative extraction over time was used to predict root extraction beyond 40 min. A maximum-likelihood approach was used to calculate confidence intervals. The temporal prediction method added 21-32% to initial estimates of standing crop root mass. According to predictions, complete manual root extraction from 18 samples would have taken c. 239 h, compared with 12 h using the prediction method. Uncertainties (percentage difference between mean, and 10th and 90th percentiles) introduced by the prediction method were small (12-15%), compared with uncertainties caused by spatial variation in root mass (72-191%, for nine samples per plot surveyed). This method provides a way of increasing the number of root samples processed per unit time, without compromising measurement accuracy.     

A comparison of methods for converting rhizotron root length measurements into estimates of root mass production per unit ground area

Rhizotrons provide valuable information about plant root production, but measurements are usually made in units of root length per unit surface area of observation window surface. These measurement units are not easily comparable to above-ground plant growth. To address this deficiency, several techniques have been developed to convert rhizotron measurement units into root mass production per unit ground area. In this study, four different conversion methods were applied to the same dataset of rhizotron measurements. This data was used to reveal the effect of conversion method upon estimates of the temporal variation in, and annual magnitude of, gross root mass production. Application of four different conversion methods resulted in gross root production estimates ranging between 2.1 and 11.4 t ha⁻¹ year⁻¹. Temporal variation in gross root mass production also varied between methods. All current methods for quantifying root production are likely to cause some disturbance and bias. Based upon a comparison of the sources of error present in each conversion method, we assess which methods are likely to produce the most reliable estimates of root biomass production per unit ground area, and propose additional measurements which could further improve accuracy.     

Oxidative folding of nerve growth factor can be mediated by the pro-peptide of neurotrophin-3

We have previously shown that the pro-peptide of human nerve growth factor (NGF) facilitates oxidative folding of the mature part. For the analysis of functional specificities of the pro-peptides of NGF and the related neurotrophin-3 (NT-3) with respect to structure formation, chimeric proteins with swapped pro-peptides were generated. Neither the structure nor the stability of the mature domains was influenced by the heterologous pro-peptides. For the pro-peptide of NT-3 fused to the mature part of NGF, stabilization of the pro-peptide moiety by the NGF part was observed. Folding kinetics and renaturation yields of this chimeric protein were comparable to those of proNGF. Our results demonstrate functional interchangeability between the pro-peptides of NGF and NT-3 with respect to their role in assisting oxidative folding of the mature part.     

A Standard Operating Procedure (SOP) for the preparation of intra- and extracellular proteins of Clostridium acetobutylicum for proteome analysis

We report on the development of a Standard Operating Procedure (SOP) for extraction and handling of intra- and extracellular protein fractions of Clostridium acetobutylicum ATCC 824 for reproducible high quality two-dimensional gel electrophoresis (2-DE) analyses. Standardized cells from a phosphate-limited chemostat were used to evaluate different protein preparation methods. For the preparation of the secretome, a dialysis/ultrafiltration procedure resulted in higher protein yields and proved to be more reliable compared to different precipitation methods using TCA, DOC-TCA, acetone, and PEG 6000. Sonication was found to be the most efficient method among different tested techniques of cell disruption for the analysis of the intracellular proteome. Furthermore, the effect of protease inhibitors and sample storage conditions were tested for both intra- and extracellular protein samples. Significant changes in the protein pattern were observed depending on the addition of protease inhibitors. 2-DE gels with a pH gradient from 4 to 7 prepared according to the developed SOP contained at least 736 intracellular and 324 extracellular protein spots.     

Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of alpha,alpha-trehalose

Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.     

Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination

V(D)J recombination events are initiated by cleavage at gene segments by the RAG1:RAG2 complex, which results in hairpin formation at the coding ends. The hairpins are opened by the Artemis:DNA-PKcs complex, and then joined via the nonhomologous DNA end joining (NHEJ) process. Here we examine the opening of the hairpinned coding ends from all of the 39 functional human V_H elements. We find that there is some sequence-dependent variation in the efficiency and even the position of hairpin opening by Artemis:DNA-PKcs. The hairpin opening efficiency varies over a 7-fold range. The hairpin opening position varies over the region from 1 to 4 nt 3′ of the hairpin tip, leading to a 2–8 nt single-stranded 3′ overhang at each coding end. This information provides greater clarity on the extent to which the hairpin opening position contributes to junctional diversification in V(D)J recombination.