Functionally different pools of Shiga toxin receptor, globotriaosyl ceramide, in HeLa cells

Many studies have investigated the intracellular trafficking of Shiga toxin, but very little is known about the underlying dynamics of its cellular receptor, the glycosphingolipid globotriaosyl ceramide. In this study, we show that globotriaosyl ceramide is required not only for Shiga toxin binding to cells, but also for its intracellular trafficking. Shiga toxin induces globotriaosyl ceramide recruitment to detergent-resistant membranes, and subsequent internalization of the lipid. The globotriaosyl ceramide pool at the plasma membrane is then replenished from internal stores. Whereas endocytosis is not affected in the recovery condition, retrograde transport of Shiga toxin to the Golgi apparatus and the endoplasmic reticulum is strongly inhibited. This effect is specific, as cholera toxin trafficking on GM(1) and protein biosynthesis are not impaired. The differential behavior of both toxins is also paralleled by the selective loss of Shiga toxin association with detergent-resistant membranes in the recovery condition, and comparison of the molecular species composition of plasma membrane globotriaosyl ceramide indicates subtle changes in favor of unsaturated fatty acids. In conclusion, this study demonstrates the dynamic behavior of globotriaosyl ceramide at the plasma membrane and suggests that globotriaosyl ceramide-specific determinants, possibly its molecular species composition, are selectively required for efficient retrograde sorting on endosomes, but not for endocytosis.     

Mutations in the inter-SH2 domain of the regulatory subunit of phosphoinositide 3-kinase: effects on catalytic subunit binding and holoenzyme function

Class IA phosphoinositide 3-kinases (PI3Ks) represent a group of heterodimeric lipid kinases with important functions in cellular signal transduction. The regulatory p85 subunit constitutively binds to the catalytic p110 subunit and mediates the recruitment of the heterodimer to various membrane-localized proteins upon activation by a vast array of stimuli. The functional characterization of protein domains that mediate p85 function has been hampered by a lack of structural data. Therefore, we investigated a 35-aa region in the inter-SH2 domain of p85, reported to be necessary for binding of p110, by site-directed mutagenesis and evaluated the importance of individual amino acids for PI3K heterodimer formation. This approach led to the identification of an 11-aa region required for p110 binding in vitro and mesoderm induction during early Xenopus development in vivo. Further analyses revealed two pairs of hydrophobic amino acids within this region, which are particularly important for high-affinity intersubunit interaction. Thus, our data provide further insight into the molecular mechanisms of PI3K intersubunit interaction and led to the identification of new p85 mutant proteins with varying degrees of dominant-negative effects that will be helpful for future PI3K-related research.     

Integrative exposure assessment through classification and regression trees on bioaccumulation of metals, related sampling site characteristics and ecoregions

The UNECE Heavy Metals in Mosses Surveys measure and spatially predict environmental concentration (PEC) of metals in mosses for ecotoxicological risk assessments. Up to now, no statistical sound investigation was dedicated to those boundary conditions which, aside from the atmospheric depositions of metals from the atmosphere down to the land surface, might influence the bioaccumulation of metals. Thus, the article focuses on the integrative analysis of the data on the bioaccumulation of metals in Germany 1990, 1995 and 2000 on the one hand and of data on conceivable boundary conditions on the other hand. To this end Classification and Regression Trees (CART) were used because CART is a very powerful statistical technique that enables to uncover structures in large data sets containing categorical and continuous data without any transformation of scale dignity. Within the framework of the Metals in Mosses Surveys, moss samples were taken at 592 sites in 1990, at 1026 sites in 1995 and at 1028 sites in 2000 in Germany. At each of them mosses were sampled and the concentrations of up to 40 metals were measured. The sampling, the ecological characteristics of the sampling sites, and the chemical analysis were documented in a metadata base. An ecoregionalisation of Germany was calculated with data on natural vegetation, altitude, soil texture and climate by means of CART. The ecoregions were mapped by GIS and intersected with multi-metal bioaccumulation indices calculated from the monitoring data 1990, 1995 and 2000. These indices were calculated by percentile statistics and the concentrations of As, Cd, Cr, Cu, Fe, Hg, Ni, Pb, V and Zn were integrated. To reach an integrative exposure assessment, for each survey a CART-model was computed encompassing the multi-metal bioaccumulation indices, the metadata and the ecoregionalization. The models describe the multivariate correlations of metal bioaccumulation with site-specific and ecoregional characteristics in a comprehensive and holistic manner over time, space and metal species. This is of crucial importance for the next step in ecological risk assessment, i.e. the interpretation of the exposure data with regard to predicted no effect concentrations (PNEC) and the sensitivity of ecosystems.     

Thermal stability of trimeric light-harvesting chlorophyll a/b complex (LHCIIb) in liposomes of thylakoid lipids

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem (PS) II functions by harvesting light energy and by limiting and balancing the energy flow directed towards the PSI and PSII reaction centers. The complex is predominantly trimeric; however, the monomeric form may play a role in one or several of the regulatory functions of LHCIIb. In this work the dissociation temperature was measured of trimeric LHCIIb isolated from Pisum thylakoids and inserted into liposomes made of various combinations of thylakoid lipids at various protein densities. Dissociation was measured by monitoring a trimer-specific circular dichroism signal in the visible range. The LHCIIb density in the membrane significantly affected the trimer dissociation temperature ranging from 70 degrees C at an LHCIIb concentration comparable to or higher than the one in thylakoid grana, to 65 degrees C at the density estimated in stromal lamellae. Omitting one thylakoid lipid from the liposomes had virtually no effect on the thermal trimer stability in most cases except when digalactosyl diacylglycerol (DGDG) was omitted which caused a drop in the apparent dissociation temperature by 2 degrees C. In liposomes containing only one lipid species, DGDG and, even more so, monogalactosyl diacylglycerol (MGDG) increased the thermal stability of LHCIIb trimers whereas phosphatidyl diacylglycerol (PG) significantly decreased it. The lateral pressure exerted by the non-bilayer lipid MGDG did not significantly influence LHCII trimer stability.     

Clinical and functional remission: even though biologics are superior to conventional DMARDs overall success rates remain low – results from RABBIT, the German biologics register

We investigated the frequency of remission according to the disease activity score (DAS28) definition, modified American Rheumatology Association (ARA) criteria, and the frequency of an achievement of a functional status above defined thresholds ('functional remission', 'physical independence') in rheumatoid arthritis (RA) patients treated with either biologics or conventional DMARDs. We used the data of a prospective cohort study, the German biologics register RABBIT (German acronym for Rheumatoid Arthritis – Observation of Biologic Therapy) to investigate the outcomes in RA patients with two or more DMARD failures who received new treatment with biologics (BIOL; n = 818) or a conventional DMARD (n = 265). Logistic regression analysis was applied to adjust for differences in baseline risks. Taking risk indicators such as previous DMARD failures or baseline clinical status into account, we found that biologics doubled the chance of remission compared to conventional DMARD therapies (DAS28 remission, adjusted odds ratio (OR) 1.95 (95% confidenece interval (CI) 1.2–3.2)); ARA remission, OR 2.05 (95% CI 1.2–3.5)). High remission rates (DAS28 remission, 30.6%; ARA remission, 16.9%) were observed in BIOL patients with a moderate disease activity (DAS28, 3.2 to 5.1) at the start of treatment. These rates decreased to 8.5% in patients with DAS28 > 6. Sustained remission at 6 and 12 months was achieved in <10% of the patients. Severely disabled patients (≤50% of full function) receiving biologic therapies were significantly more likely to achieve a status indicating physical independence (≥67% of full function) than controls (OR 3.88 (95% CI 1.7–8.8)). 'Functional remission' (≥83% of full function) was more often achieved in BIOL than in controls (OR 2.18 (95% CI 1.04–4.6)). In conclusion, our study shows that biologics increase the chance to achieve clinical remission and a status of functional remission or at least physical independence. However, temporary or even sustained remission remain ambitious aims, which are achieved in a minority of patients only.     

Variation of particulate carbohydrate pools over time and depth in a diatom-dominated plankton community at the Antarctic Polar Front

Carbohydrate dynamics were studied in an algal community dominated by the diatom Fragilariopsis kerguelensis at the Antarctic Polar Front during austral autumn 1999. Water-extractable mono- and polysaccharide concentrations from the particulate fraction were measured at six depths in the upper 100 m at seven stations along a N-S transect (20°E, 48-50°S) during 2 consecutive days. In addition, field populations were incubated on deck for 18 h at four different light intensities. Polysaccharide concentrations varied between 2.7 and 13.6 wg/l, monosaccharide concentrations between 2.5 and 8.9 wg/l. Near the surface (0-60 m), a diel pattern was observed in the polysaccharide concentration when normalised to the chlorophyll a or the monosaccharide concentration. The deck incubations supported the hypothesis that this pattern resulted from the diurnal accumulation and nocturnal consumption of reserve glucan. More polysaccharides accumulated at high light intensities than at low light intensities, in accordance with the observed decrease in normalised polysaccharide concentration with depth. In addition, the lower concentrations at depth might be explained by consumption: polysaccharides that were accumulated during time spent near the surface were subsequently respired when cells were transported to deeper and dimmer water layers. The variation in carbohydrate pools over time and depth described here must be considered of ecological relevance to phytoplankton in the Southern Ocean subjected to extended periods of darkness (hours to days) due to vertical mixing and advection.     

Proteomic‐based investigations on the mode of action of the marine anticancer compound rhizochalinin

Rhizochalinin (Rhiz) is a novel marine natural sphingolipid‐like compound, which shows promising in vitro and in vivo activity in human castration‐resistant prostate cancer. In the present study, a global proteome screening approach was applied to investigate molecular targets and biological processes affected by Rhiz in castration‐resistant prostate cancer. Bioinformatical analysis of the data predicted an antimigratory effect of Rhiz on cancer cells. Validation of proteins involved in the cancer‐associated processes, including cell migration and invasion, revealed downregulation of specific isoforms of stathmin and LASP1, as well as upregulation of Grp75, keratin 81, and precursor IL‐1β by Rhiz. Functional analyses confirmed an antimigratory effect of Rhiz in PC‐3 cells. Additionally, predicted ERK1/2 activation was confirmed by Western blotting analysis, and revealed prosurvival effects in Rhiz‐treated prostate cancer cells indicating a potential mechanism of resistance. A combination of Rhiz with MEK/ERK inhibitors PD98059 (non‐ATP competitive MEK1 inhibitor) and FR180204 (ATP‐competitive ERK1/2 inhibitor) resulted in synergistic effects. This work provides further insights into the molecular mechanisms underlying Rhiz bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anticancer agents.