Effects of shrub encroachment on the anuran community in periodically flooded grasslands of the largest Neotropical wetland

In many parts of the world, replacement of natural grasslands by woody plants has resulted in a decrease of pasture areas and in habitat loss for a variety of animal species, including amphibians. Wetlands are especially susceptible to invasive plants, both native and exotic, but the effects of such invasions on animal assemblages remain poorly understood. Here, we present information on the impact of selected environmental variables, especially coverage by the native shrub Combretum laxum Jacq., on the structure of an anuran assemblage in the Pantanal, a huge flood‐pulsed South American wetland. Anurans were surveyed during the rainy season in 17 plots, which differed in extent of C. laxum coverage, leaf litter volume, soil moisture and distance to permanently wet areas. Effects of these environmental variables on the species number, relative abundance and composition of the anuran assemblage were evaluated using multivariate statistical analyses. We captured 1203 anurans, of 21 species from four families. Both the number of species and the relative abundance of anurans were lower in plots with greater C. laxum coverage, which also influenced anuran species composition. Number of species was highest in plots located closest to permanently wet areas, which provide protection from desiccation and other resources during the Pantanal dry season, and so could be considered source areas of anurans. While many anuran species were negatively affected by the homogenization of the landscape caused by shrub encroachment, some seemed to be favoured in such circumstances. For these, dense shrub encroachment into natural grasslands may provide safer migratory routes to permanently wet habitats. Thus, at the mesoscale, a mosaic of areas with different levels of coverage by C. laxum (shrub islands) may aid anuran assemblages in the Pantanal wetlands, facilitating the maintenance of higher beta and gamma diversity.     

Fracture prevention in COPD patients; a clinical 5-step approach

Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV₁ < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture.     

Evidence for cadherin-11 cleavage in the synovium and partial characterization of its mechanism

IntroductionEngagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. The objective of this study was to determine if cadherin-11 extracellular domain fragments are found inside the joint and if a physiologic synovial fibroblast cleavage pathway releases those fragments.MethodsCadherin-11 cleavage fragments were detected by western blot in cell media or lysates. Cleavage was interrupted using chemical inhibitors or short-interfering RNA (siRNA) gene silencing. The amount of cadherin-11 fragments in synovial fluid was measured by western blot and ELISA.ResultsSoluble cadherin-11 extracellular fragments were detected in human synovial fluid at significantly higher levels in RA samples compared to osteoarthritis (OA) samples. A cadherin-11 N-terminal extracellular binding domain fragment was shed from synovial fibroblasts after ionomycin stimulation, followed by presenilin 1 (PSN1)-dependent regulated intramembrane proteolysis of the retained membrane-bound C-terminal fragments. In addition to ionomycin-induced calcium flux, tumor necrosis factor (TNF)-α also stimulated cleavage in both two- and three-dimensional fibroblast cultures. Although cadherin-11 extracellular domains were shed by a disintegrin and metalloproteinase (ADAM) 10 in several cell types, a novel ADAM- and metalloproteinase-independent activity mediated shedding in primary human fibroblasts.ConclusionsCadherin-11 undergoes ectodomain shedding followed by regulated intramembrane proteolysis in synovial fibroblasts, triggered by a novel sheddase that generates extracelluar cadherin-11 fragments. Cadherin-11 fragments were enriched in RA synovial fluid, suggesting they may be a marker of synovial burden and may function to modify cadherin-11 interactions between synovial fibroblasts.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0647-9) contains supplementary material, which is available to authorized users.     

Immunolocalization of IFN-gamma in the lesions of resistant and susceptible mice to Paracoccidioides brasiliensis infection

The important role of interferon-gamma (IFN-γ) in protective immunity in mycosis is well established, except for its participation in fungal granulomas. Herein, we employ immunohistochemical reactions to describe the in situ localization of IFN-γ in granulomas of susceptible (B10.A) and resistant (A/J) mice to infection with Paracoccidioides brasiliensis (Pb). After infection with the highly virulent Pb18, IFN-γ-positive lymphomononuclear cells were localized mainly at the periphery of granulomas in both mouse strains. The numbers of positive cells found in compact granulomas of A/J mice increased significantly from 15 to 120 days postinfection. At this time, significantly more positive cells were detected in the compact granulomas of resistant mice than in the loose, multifocal lesions of the susceptible ones. In infection with the slightly virulent Pb265, the same pattern of IFN-γ localization was found as in Pb18 infection, but there was decreased staining at 120 days due to the presence of only residual lesions in both mouse strains. The marked IFN-γ staining observed in the granulomas of resistant mice at the later stage of Pb infection confirms its importance in fungal dissemination control, and suggests a contribution to the development of paracoccidioidal granuloma.     

Expression of functional recombinant human growth hormone in transgenic soybean seeds

We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α') subunit of β-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9?g?kg(-1)) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.     

Synthesis of new glycyrrhetinic acid derived ring A azepanone, 29-urea and 29-hydroxamic acid derivatives as selective 11β-hydroxysteroid dehydrogenase 2 inhibitors

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2. Whereas inhibition of 11β-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11β-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. Recently, we published a series of hydroxamic acid derivatives of glycyrrhetinic acid showing high selectivity for 11β-HSD2. The most potent and selective compound is active against human 11β-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11β-HSD1. Starting from the lead compounds glycyrrhetinic acid and the hydroxamic acid derivatives, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11β-HSD1 and 11β-HSD2 in cell lysates. Here we describe novel 29-urea- and 29-hydroxamic acid derivatives of glycyrrhetinic acid as well as derivatives with the Beckman rearrangement of the 3-oxime to a seven-membered ring, and the rearrangement of the C-ring from 11-keto-12-ene to 12-keto-9(11)-ene. The combination of modifications on different positions led to compounds comprising further improved selective inhibition of 11β-HSD2 in the lower nanomolar range with up to 3600-fold selectivity.     

Heat-Induced Whey Protein Gels: Effects of pH and the Addition of Sodium Caseinate

The effects of pH (6.7 or 5.8), protein concentration and the heat treatment conditions (70 or 90 °C) on the physical properties of heat-induced milk protein gels were studied using uniaxial compression, scanning electron microscopy, differential scanning calorimetry, and water-holding capacity measurements. The systems were formed from whey protein isolate (10–15% w/v) with (5% w/v) or without the addition of caseinate. The reduction in pH from 6.7 to 5.8 increased the denaturation temperature of the whey proteins, which directly affected the gel structure and mechanical properties. Due to this increase in the denaturation temperature of the β-lactoglobulin and α-lactalbumin, a heat treatment of 70 °C/30 min did not provide sufficient protein unfolding to form self-supporting gels. However, the presence of 5% (w/v) sodium caseinate decreased the whey protein thermo stability and was essential for the formation of self-supporting gels at pH 6.7 with heat treatment at 70 °C/30 min. The gels formed at pH 6.7 showed a fine-stranded structure, with great rigidity and deformability as compared to those formed at pH 5.8. The latter had a particulate structure and exuded water, which did not occur with the gels formed at pH 6.7. The addition of sodium caseinate led to less porous networks with increased gel deformability and strength but decreased water exudation. The same tendencies were observed with increasing whey protein concentration.     

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

Pancreatic β-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of β-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in β-cells following exposure to well-defined β-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the β-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to β-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.     

Chemosymbiotic bivalves from the mud volcanoes of the Gulf of Cadiz, NE Atlantic, with descriptions of new species of Solemyidae, Lucinidae and Vesicomyidae

The chemosymbiotic bivalves collected from the mud volcanoes of the Gulf of Cadiz are reviewed. Of the thirteen species closely associated with chemosynthetic settings two Solemyidae, Solemya (Petrasma) elarraichensissp. n. and Acharax gadiraesp. n., one Lucinidae, Lucinoma asapheussp. n., and one Vesicomyidae, Isorropodon megadesmussp. n. are described and compared to close relatives of their respective families. The biodiversity and distribution of the chemosymbiotic bivalves in the Gulf of Cadiz are discussed and compared to the available information from other cold seeps in the Eastern Atlantic and Mediterranean. Although there is considerable similarity at the genus level between seep/mud volcano fields in the Eastern Atlantic and Mediterranean, there is little overlap at the species level. This indicates a high degree of endemism within chemosymbiotic bivalve assemblages.     

Production and characterization of a collagenolytic serine proteinase by <Emphasis Type="Italic">Penicillium aurantiogriseum</Emphasis> URM 4622: A factorial study

A 2⁴ full factorial design was used to identify the main effects and interactions of the initial medium pH, soybean flour concentration, temperature and orbital agitation speed on extracellular collagenase production by Penicillium aurantiogriseum URM4622. The most significant variables for collagenase production were soybean flour concentration and initial medium pH that had positive main effects, and temperature that had a negative one. Protein concentration in soybean flour revealed to be a significant factor for the production of a collagenase serine proteinase. The most favorable production conditions were found to be 0.75% soybean flour, pH 8.0, 200 rpm, and 28°C, which led to a collagenase activity of 164 U. The enzyme showed an optimum activity at 37°C and pH 9.0, was stable over wide ranges of pH and temperature (6.0 ∼ 10.0 and 25 ∼ 45°C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonylfluoride. The firstorder rate constants for collagenase inactivation in the crude extract, calculated from semi-log plots of the residual activity versus time, were used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* _d = 107.4 kJ/mol and ΔH* _d = 104.7 kJ/mol). The enzyme is probably an extracellular neutral serine collagenase effective on azocoll, gelatin and collagen decomposition.