Distribution of Hepatotoxic Cyanobacterial Blooms in Belgium and Luxembourg

A survey of the distribution of cyanobacterial blooms in the southern part of Belgium, in Luxembourg as well as in bordering northeastern France was carried out for 4 years (1997, 1999–2001). In the 64 cyanobacterial bloom samples collected, Microcystis as well as Planktothrix were the most frequently encountered dominant bloom formers, followed by Anabaena, Woronichinia, and Aphanizomenon. The relative frequency of (co-)dominant genera was highly correlated to the geology of the catchments. Microcystins were found in 53% of the analysed blooms and their presence was mainly assigned to Microcystis dominance. The highest microcystin concentration of 2231 μg g⁻¹ seston DW was recorded in a sample dominated by Woronichinia naegeliana. Among the 6 investigated microcystin variants, MC-LR was the most frequently detected whereas MC-LY was never revealed.     

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.     

Olfactory and lens placode formation is controlled by the hedgehog-interacting protein (Xhip) in Xenopus

The integration of multiple signaling pathways is a key issue in several aspects of embryonic development. In this context, extracellular inhibitors of secreted growth factors play an important role, which is to antagonize specifically the activity of the corresponding signaling molecule. We provide evidence that the Hedgehog-interacting protein (Hip) from Xenopus, previously described as a Hedgehog-specific antagonist in the mouse, interferes with Wnt-8 and eFgf/Fgf-8 signaling pathways as well. To address the function of Hip during early embryonic development, we performed gain- and loss-of-function studies in the frog. Overexpression of Xhip or mHip1 resulted in a dramatic increase of retinal structures and larger olfactory placodes primarily at the expense of other brain tissues. Furthermore, loss of Xhip function resulted in a suppression of olfactory and lens placode formation. Therefore, the localized expression of Xhip may counteract certain overlapping signaling activities, which inhibit the induction of distinct sensory placodes.     

Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking

Tubules and vesicles are membrane carriers involved in traffic along the endocytic and secretory routes. The small GTPase Arf6 regulates a recycling branch of short dynamic tubular intermediates used by major histocompatibility class I (MHC-I) molecules to traffic through vesicles between endosomes and the plasma membrane. We observed that Arf6 also affects a second network of very long and stable tubules containing MHC-I, many of which correspond to deep invaginations of the plasma membrane. Treatment with wortmannin, an inhibitor of phosphatidylinositol-3-phosphate kinase, prevents formation of the short dynamic tubules while increasing the number of the long and very stable ones. Expression of NefAAAA, a mutant form of HIV Nef, increases the number of cells containing the stable tubules, and is used here as a tool to facilitate their study. Photoactivation of NefAAAA-PA-GFP demonstrates that this molecule traffics from endosomes to the tubules. Finally, live-cell imaging also shows internalization of MHC-I molecules into these tubules, suggesting that this is an additional route for MHC-I traffic.     

Endocytosis is not required for the selective lipid uptake mediated by murine SR-BI

The scavenger receptor class B, type I (SR-BI) mediates the cellular selective uptake of cholesteryl esters and other lipids from high-density lipoproteins (HDL) and low-density lipoproteins (LDL). This process, unlike classical receptor-mediated endocytosis, does not result in lipoprotein degradation. Instead, the lipid depleted particles are released into the medium. Here we show that selective lipid uptake mediated by murine SR-BI can be uncoupled from the endocytosis of HDL or LDL particles. We found that blocking selective lipid uptake by incubating cells with the small chemical inhibitors BLT-1 or BLT-4 did not affect endocytosis of HDL. Similarly, blocking endocytosis by hyperosmotic sucrose or K+ depletion did not prevent selective lipid uptake from HDL or LDL. These findings suggest that mSR-BI-mediated selective uptake occurs at the cell surface upon the association of lipoproteins with mSR-BI and does not require endocytosis of HDL or LDL particles.     

Electroimmobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purification

Proinsulin C-peptide was electroimmobilized to a quartz crystal microbalance sensor chip, localizing this low-pI peptide for covalent attachment to activated surface carboxyl groups. The resulting chip was used in a continuous flow biosensor to capture anti-C-peptide antibodies, which could subsequently be eluted in 5% formic acid between air bubbles for efficient recovery and mass spectrometric identification. The method is reproducible through repeated cycles, providing affinity purification of proteins under real-time monitoring of the binding and elution processes.     

Determination of site-specificity of S-glutathionylated cellular proteins

Redox modification by S-glutathionylation is an expanding field within cell signalling research. However, the methods available for analysis of S-glutathionylated proteins in complex mixtures are not sufficiently accurate to specifically and in a high-throughput manner on a structural level establish the effects of S-glutathionylation on the individual proteins. A method has been developed for rapid identification of the S-glutathionylation sites of proteins in diamide-treated ECV304 cells, through tagging of deglutathionylated proteins with a cysteine-reactive biotin-affinity tag, trypsinisation, avidin-affinity purification of tagged peptides, and subsequent analysis by liquid chromatography and quadrupole time-of-flight tandem mass spectrometry. The method has led to identification of the glutathionylation sites of gamma-actin (Cys(217)), heat shock protein 60 (Cys(447)), and elongation factor 1-alpha-1 (Cys(411)). Further developments of accuracy within the field of peptide-affinity capture and mass spectrometry are discussed.