Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation

T(H)17 lymphocytes appear to be essential in the pathogenesis of numerous inflammatory diseases. We demonstrate here the expression of IL-17 and IL-22 receptors on blood-brain barrier endothelial cells (BBB-ECs) in multiple sclerosis lesions, and show that IL-17 and IL-22 disrupt BBB tight junctions in vitro and in vivo. Furthermore, T(H)17 lymphocytes transmigrate efficiently across BBB-ECs, highly express granzyme B, kill human neurons and promote central nervous system inflammation through CD4+ lymphocyte recruitment.     

Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.     

Localization of ORC1 during the cell cycle in human leukemia cells

The interaction of the origin recognition complex (ORC) with replication origins is a critical parameter in eukaryotic replication initiation. In mammals the ORC remains bound except during mitosis, thus the localization of ORC complexes allows localization of origins. A monoclonal antibody that recognizes human ORC1 was used to localize ORC complexes in populations of human MOLT-4 cells separated by cell cycle position using centrifugal elutriation. ORC1 staining in cells in early G1 is diffuse and primarily peripheral. As the cells traverse G1, ORC1 accumulates and becomes more localized towards the center of the nucleus, however around the G1/S boundary the staining pattern changes and ORC1 appears peripheral. By mid to late S phase ORC1 immunofluorescence is again concentrated at the nuclear center. During anaphase, ORC1 staining is localized mainly in the pericentriolar regions. These findings suggest that concerted movements of origin DNA sequences in addition to the previously documented assembly and disassembly of protein complexes are an important aspect of replication initiation loci in eukaryotes.     

Double standards in quantitative proteomics: direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling

Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. In this report we evaluate in a direct comparative assessment two state-of-the-art quantitative proteomic approaches, namely difference in gel electrophoresis (DiGE) and metabolic stable isotope labeling. Therefore, Saccharomyces cerevisiae was grown under well defined experimental conditions in chemostats under two single nutrient-limited growth conditions using (14)N- or (15)N-labeled ammonium sulfate as the single nitrogen source. Following lysis and protein extraction from the two yeast samples, the proteins were fluorescently labeled using different fluorescent CyDyes. Subsequently, the yeast samples were mixed, and the proteins were separated by two-dimensional gel electrophoresis. Following in-gel digestion, the resulting peptides were analyzed by mass spectrometry using a MALDI-TOF mass spectrometer. Relative ratios in protein expression between these two yeast samples were determined using both DiGE and metabolic stable isotope labeling. Focusing on a small, albeit representative, set of proteins covering the whole gel range, including some protein isoforms and ranging from low to high abundance, we observe that the correlation between these two methods of quantification is good with the differential ratios determined following the equation R(Met.Lab.) = 0.98R(DiGE) with r(2) = 0.89. Although the correlation between DiGE and metabolic stable isotope labeling is exceptionally good, we do observe and discuss (dis)advantages of both methods as well as in relation to other (quantitative) approaches.     

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.     

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.     

Iron content of sediment and phosphate adsorption properties

Phosphorus can occur in sediments in different forms and accordingly its availability varies. The distinction between the phosphorus fractions is made with two chemical extraction methods; an ammonium oxalate-oxalic acid extraction and an extraction according to Hieltjes & Lijklema (1980).The iron and aluminum liberated with the ammonium oxalate-oxalic acid extraction method is linearly correlated (r ² = 0.73) with the phosphorus liberated in the first two steps of the Hieltjes and Lijklema extraction by: P = 0.035 (Fe + Al) + 0.001 (P, Fe and Al in mmol g^–1).The iron and aluminum (hydr)oxides are very important fractions in the sediment adsorption capacity for phosphorus. The phosphorus sorption capacity (PSC) is 0.080 mol P (mol (Fe + Al))^–1 and the adsorption constant (k) is 11.9 µmol P l^–1. Here it is assumed that iron and aluminum (hydr)oxide have the same affinity for phosphorus.     

Temporal variations and pond age effect on plankton communities in semi-intensive freshwater marron (Cherax cainii,Austin and Ryan, 2002) earthen aquaculture ponds in Western Australia

The abundance and diversity of the plankton community represents the health of the aquatic ecosystem, and plays an important role in the growth of cultured animals under aquaculture conditions. The temporal variations of plankton abundance, taxonomic composition, diversity, evenness and species richness were studied in three old and three new semi-intensive marron (Cherax cainii, Austin and Ryan, 2002) ponds. Water parameters such as temperature, dissolved oxygen, pH, turbidity, TAN, nitrite, nitrate and reactive phosphate were recorded, and plankton samples were collected every two months, for one year of juvenile production cycle. A total of twenty-six phytoplankton and seven zooplankton genera were recorded. Chlorophyceae was the dominant class of phytoplankton throughout the year, followed by Trebouxiophyceae. Rotifera comprised 49.8% of the total zooplankton community (individuals L^?1), the largest proportion of any group. Temporal variations impacted the plankton abundance and community structure, and plankton abundance were more abundant during summer. The pond age did not influence the phytoplankton abundance, whereas zooplankton abundance was higher in older ponds.