Synergism between curdlan and GM-CSF confers a strong inflammatory signature to dendritic cells

A simultaneous engagement of different pathogen recognition receptors provides a tailor-made adaptive immunity for an efficient defense against distinct pathogens. For example, cross-talk of TLR and C-type lectin signaling effectively shapes distinct gene expression patterns by integrating the signals at the level of NF-κB. In this study, we extend this principle to a strong synergism between the dectin-1 agonist curdlan and an inflammatory growth factor, GM-CSF. Both together act in synergy in inducing a strong inflammatory signature that converts immature dendritic cells (DCs) to potent effector DCs. A variety of cytokines (IL-1β, IL-6, TNF-α, IL-2, and IL-12p70), costimulatory molecules (CD80, CD86, CD40, and CD70), chemokines (CXCL1, CXCL2, CXCL3, CCL12, CCL17), as well as receptors and molecules involved in fugal recognition and immunity such as Mincle, dectin-1, dectin-2, and pentraxin 3 are strongly upregulated in DC treated simultaneously with curdlan and GM-CSF. The synergistic effect of both stimuli resulted in strong IκBα phosphorylation, its rapid degradation, and enhanced nuclear translocation of all NF-κB subunits. We further identified MAPK ERK as one possible integration site of both signals, because its phosphorylation was clearly augmented when curdlan was coapplied with GM-CSF. Our data demonstrate that the immunomodulatory activity of curdlan requires an additional signal provided by GM-CSF to successfully initiate a robust β-glucan-specific cytokine and chemokine response. The integration of both signals clearly prime and tailor a more effective innate and adaptive response against invading microbes and fungi.     

Total antioxidant performance: a validated fluorescence assay for the measurement of plasma oxidizability

The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750mug/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, alpha-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid (R=0.97, P<0.05) and cholesterol-adjusted alpha-tocopherol (R=0.93, P<0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.     

Uses,traditional management,perception of variation and preferences in ackee (Blighia sapida K.D. Koenig) fruit traits in Benin: implications for domestication and conservation

Background

Changing lifestyles have recently caused a severe reduction of the gathering of wild food plants. Knowledge about wild food plants and the local environment becomes lost when plants are no longer gathered. In Central Europe popular scientific publications have tried to counter this trend. However, detailed and systematic scientific investigations in distinct regions are needed to understand and preserve wild food uses. This study aims to contribute to these investigations.

Methods

Research was conducted in the hill country east of Graz, Styria, in Austria. Fifteen farmers, most using organic methods, were interviewed in two distinct field research periods between July and November 2008. Data gathering was realized through freelisting and subsequent semi-structured interviews. The culinary use value (CUV) was developed to quantify the culinary importance of plant species. Hierarchical cluster analysis was performed on gathering and use variables to identify culture-specific logical entities of plants. The study presented was conducted within the framework of the master's thesis about wild plant gathering of the first author. Solely data on gathered wild food species is presented here.

Results

Thirty-nine wild food plant and mushroom species were identified as being gathered, whereas 11 species were mentioned by at least 40 percent of the respondents. Fruits and mushrooms are listed frequently, while wild leafy vegetables are gathered rarely. Wild foods are mainly eaten boiled, fried or raw. Three main clusters of wild gathered food species were identified: leaves (used in salads and soups), mushrooms (used in diverse ways) and fruits (eaten raw, with milk (products) or as a jam).

Conclusions

Knowledge about gathering and use of some wild food species is common among farmers in the hill country east of Graz. However, most uses are known by few farmers only. The CUV facilitates the evaluation of the culinary importance of species and makes comparisons between regions and over time possible. The classification following gathering and use variables can be used to better understand how people classify the elements of their environment. The findings of this study add to discussions about food heritage, popularized by organizations like Slow Food, and bear significant potential for organic farmers.     

Modification of host lipid raft proteome upon hepatitis C virus replication

Hepatitis C virus (HCV) replication complex resides in detergent-insoluble subcellular domains or lipid rafts. We used two proteomics approaches to characterize the protein content of lipid rafts isolated from Huh7 cells and its modification upon HCV replication. Using two-dimensional electrophoresis and mass spectrometry, we identified approximately 100 protein spots in the isolated lipid rafts; among them, 39 were reproducibly modified in HCV replicon cell lines as compared with control cell lines. We also used stable isotope labeling by amino acids in cell culture (SILAC) combined with one-dimensional electrophoresis separation and mass spectrometry. Using this approach, we identified 1036 individual proteins based on peptides selected with at least 95% confidence; among them, 413 proteins were identified with at least two peptides. Quantification analysis identified 150 proteins modified by at least 2.5-fold (110 up-regulated and 40 down-regulated) in HCV-replicating cells compared with controls. Protein identifications and quantifications obtained by both proteomics approaches were largely concordant. Modulated proteins included a majority of proteins involved in vesicular and protein trafficking and in cell signaling. Remarkably for a large number of proteins, their up-regulation in lipid rafts of HCV replicon cells was due to their relocalization. By using small interfering RNAs directed to the modulated small GTPases Cdc42 and RhoA, we observed an increase in HCV replication, whereas reduction of syntaxin 7 expression resulted in decreased replication of HCV. Our findings indicate that protein subcellular relocalization occurs in HCV-containing cells that can directly affect HCV replication.     

Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation

Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway.     

Interaction model for anthracycline activity against DNA topoisomerase II

DNA topoisomerase II (Top2) is an essential nuclear enzyme and a target of very effective anticancer drugs including anthracycline antibiotics. Even though several aspects of drug activity against Top2 are understood, the drug receptor site is not yet known. Several Top2 mutants have altered drug sensitivity and have provided information of structural features determining drug action. Here, we have investigated the sensitivity to three closely related anthracycline derivatives of yeast Top2 bearing mutations in the CAP-like domain and integrated the findings with computer models of ternary drug-enzyme-DNA complexes. The results suggest a model for the anthracycline receptor wherein a drug molecule has specific interactions with the cleaved DNA as well as amino acid residues of the CAP-like domain of an enzyme monomer. The drug molecule is intercalated into DNA at the site of cleavage, and interestingly, drug-enzyme contacts involve one side of the four-ring chromophore and the side chain of the anthracycline molecule. The findings may explain several established structure-activity relationships of antitumor anthracyclines and may thus provide a framework for further developments of effective Top2 poisons.     

Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys

Using the strictly neutral model as a null hypothesis, we tested for deviations from expected levels of nucleotide polymorphism at the alcohol dehydrogenase locus (Adh-1) within and among four species of pocket gophers (Geomys bursarius major, G. knoxjonesi, G. texensis llanensis, and G. attwateri). The complete protein-encoding region was examined, and 10 unique alleles, representing both electromorphic and cryptic alleles, were used to test hypotheses (e.g., the neutral model) concerning the maintenance of genetic variation. Nineteen variable sites were identified among the 10 alleles examined, including 9 segregating sites occurring in synonymous positions and 10 that were nonsynonymous. Several statistical methods, including those that test for within-species variation as well as those that examine variation within and among species, failed to reject the null hypothesis that variation (both within and between species of Geomys) at the Adh locus is consistent with the neutral theory. However, there was significant heterogeneity in the ratio of polymorphism to divergence across the gene, with polymorphisms clustered in the first half of the coding region and fixed differences clustered in the second half of the gene. Two alternative hypotheses are discussed as possible explanations for this heterogeneity: an old balanced polymorphism in the first half of the gene or a recent selective sweep in the second half of the gene.      

Seasonal recruitment,habitat associations and survival of pomacentrid reef fish in the US Virgin Islands

Patterns of distribution and abundance of coral reef fish depend in part on recruitment of a pelagic larval stage, on subsequent dispersal among habitats, and survival of new recruits. We studied recruitment of five species of Stegastes and two species of Chromis damselfish onto reef habitats of St. Thomas, USVI during one year. The two study sites, Flat Cay and Outer Brass Island, were on the southern and northern sides of St. Thomas, respectively. At both sites, recruitment occurred largely in the summer months, although one species (Stegastes planifrons) showed significant winter recruitment at Flat Cay. The onset of increased summer recruitment in 1992 of other species occurred several weeks later and was shorter in duration at Outer Brass Island than at Flat Cay, perhaps indicating differences in oceanographic conditions (currents etc.) or spawning cycles between sites. The two Chromis species showed lunar periodicity of settlement at Flat Cay. At Flat Cay, recruits of three species (S. leucostictus, S. diencaeus and S. planifrons) were associated with conspecifics possibly due to preferential settlement. Similarly, new recruits were more often found near live coral than coral rubble, and very few occurred on sand habitat. Substratum complexity was a poor predictor of recruitment within a habitat, although larger juveniles of some species were more common on more complex substrate. Contrary to other studies, there were no apparent depth preferences among recruits, although larger juveniles of two Stegastes species were found more often in deeper water. It appears that within habitats, newly arriving larvae may be attracted first to the presence of conspecifics and secondarily take up position adjacent to live coral. Apparent survivorship of some Stegastes species and one Chromis species was higher at Outer Brass Island than at Flat Cay, and may partly compensate for lower recruitment of some species at Outer Brass Island.