Carbohydrates in the North Sea during spring blooms of <i>Phaeocystis</i>: a specific fingerprint

Regional and temporal variation in the composition of water-soluble carbohydrates from Phaeocystis colonies sampled in the southern North Sea was small during spring 1994, except for a high variability in the contribution of glucose. Glucose is universally present in storage products of microalgae; the relative constancy of the carbohydrate pattern of the other monosaccharides suggests that these are part of the more refractory colony mucus. In all Phaeocystis samples arabinose dominated, followed by xylose (Belgian coast) or galactose and mannose (Dutch coast). Rhamnose, glucuronate and O-methylated sugars were present in lower amounts. The latter, always present in samples containing Phaeocystis, may be typical for North Sea strains. The sugar patterns we report here differ from those presented in the literature concerning Phaeocystis-derived material, and also from the sugar fingerprint in the preceding diatom bloom. The Phaeocystis mucus apparently behaves as particulate matter since it was retained on filters of over 1 um. This characteristic together with its refractory nature, typical of 'transparent exopolymer particles' (TEPs), must have consequences for the heterotrophic microbial community in terms of adherence and substrate availability

Evidence for inhibition of bacterial luminescence by allelochemicals from Fibrocapsa japonica (Raphidophyceae), and the role of light and microalgal growth rate

The marine microalga Fibrocapsa japonica Toriumi and Takano (Raphidophyceae) produces haemolysins, neurotoxins and reactive oxygen species (ROS). To quantify potential effects of such bioactive compounds on surrounding organisms the marine bacterium Vibrio fischeri was exposed to F. japonica culture samples. Inhibition of V. fischeri 's natural luminescence, indicative of impaired metabolism, was related to the number of F. japonica cells added. The effect was fast, within 15 min. It was caused by one, possibly several, excreted substances that were less active after heating. Freezing of culture supernatant partly inactivated these substances, but ROS-scavenging enzymes had no effect. Light enhanced the V. fischeri luminescence inhibition in two ways. The direct effect of light on the action of F. japonica luminescence inhibiter(s) could be described by a saturation curve with maximum effect above 20 mu mol photons m(-2) s(-1). Light also had an indirect effect: biomass production, dependent on light availability, was closely related to the amount of inhibiting compound(s) produced by the alga. Algal growth rate, rather than its cell density, determined the bacterial luminescence inhibition per F. japonica cell, resulting in a 5-fold stronger inhibition at maximum growth rates compared to cells that barely grew during the stationary growth phase. The bioassay with F. japonica and V. fischeri has allowed quantification of the negative effects on bacteria in the microalgal microenvironment. The results presented here suggest that at favourable growth conditions F. japonica releases bioactive compounds that improve its competitive abilities

High acrylate concentrations in the mucus of Phaeocystis globosa colonies

Acrylate produced from dimethylsulphoniopropionate (DMSP) by Phaeocystis has been claimed to inhibit bacterial growth. However, the concentrations of acrylate measured in seawater during Phaeocystis blooms are not high enough to expect inhibition of bacterial growth. In this study, the total acrylate in Phaeocystis cultures free from bacteria was measured. The concentration found in the exponential phase of growth was similar (0.1 to 1.0 mu M) to earlier field reports, but the amount found in the stationary phase of growth was much higher (1 to 4 mu M). Acrylate in cultures, as well as in field samples. was found to be located in the mucous layer of the colony. 'Microscale' concentrations in that layer were more than 1000-fold higher (1.3 to 6.5 mM) than the total concentration found in the unfractionated culture. Such high concentrations could have an antimicrobial effect. However, acrylate appears to be adsorbed to the mucus and may be inaccessible to bacteria. including those that consume acrylate. As soon as the colonies started to decay, acrylate was released into the surrounding environment, and since it is not detected in bloom samples, it is apparently consumed by bacteria

Carba-Cyclophellitols are Neutral Retaining Glucosidase Inhibitors

The conformational analysis of glycosidases affords a route to their specific inhibition through transition-state mimicry. Inspired by the rapid reaction rates of cyclophellitol and cyclophellitol aziridineboth covalent retaining β-glucosidase inhibitorswe postulated that the corresponding carba “cyclopropyl” analogue would be a potent retaining β-glucosidase inhibitor for those enzymes reacting through the ⁴H₃ transition-state conformation. <i>Ab initio</i> metadynamics simulations of the conformational free energy landscape for the cyclopropyl inhibitors show a strong bias for the ⁴H₃ conformation, and carba-cyclophellitol, with an <i>N</i>-(4-azidobutyl)­carboxamide moiety, proved to be a potent inhibitor (<i>K</i>_i = 8.2 nM) of the <i>Thermotoga maritima</i> <i>Tm</i>GH1 β-glucosidase. 3-D structural analysis and comparison with unreacted epoxides show that this compound indeed binds in the ⁴H₃ conformation, suggesting that conformational strain induced through a cyclopropyl unit may add to the armory of tight-binding inhibitor designs

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls

Human B Cells Engage the NCK/PI3K/RAC1 Axis to Internalize Large Particles via the IgM-BCR

Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles