Synthesis, physico-chemical properties and complexing abilities of new amphiphilic ligands from d-galacturonic acid

This paper describes a convenient and efficient synthesis of new complexing surfactants from d-galacturonic acid and n-octanol as renewable raw materials in a two-step sequence. In the first step, simultaneous O-glycosidation-esterification under Fischer conditions was achieved. The anomeric ratio of the products was studied based on the main experimental parameters and the activation mode (thermal or microwave). In the second step, aminolysis of the n-octyl ester was achieved with various functionalized primary amines under standard thermal or microwave activation. The physico-chemical properties of these new amphiphilic ligands were measured and these compounds were found to exhibit interesting surface properties. Complexing abilities of one uronamide ligand functionalized with a pyridine moiety toward Cu(II) ions was investigated in solution by EPR titrations. A solid compound was also synthesized and characterized, its relative structure was deduced from spectroscopic data.     

Polyene macrolide antifungal drugs trigger interleukin-1β secretion by activating the NLRP3 inflammasome

The use of antimycotic drugs in fungal infections is based on the concept that they suppress fungal growth by a direct killing effect. However, amphotericin and nystatin have been reported to also trigger interleukin-1β (IL-1β) secretion in monocytes but the molecular mechanism is unknown. Here we report that only the polyene macrolides amphotericin B, nystatin, and natamycin but none of the tested azole antimycotic drugs induce significant IL-1β secretion in-vitro in dendritic cells isolated from C57BL/6 mouse bone marrow. IL-1β release depended on Toll-like receptor-mediated induction of pro-IL-1β as well as the NLRP3 inflammasome, its adaptor ASC, and caspase-1 for enzymatic cleavage of pro-IL-1β into its mature form. All three drugs induced potassium efflux from the cells as a known mechanism for NLRP3 activation but the P2X7 receptor was not required for this process. Natamycin-induced IL-1β secretion also involved phagocytosis, as cathepsin activation as described for crystal-induced IL-1β release. Together, the polyene macrolides amphotericin B, nystatin, and natamycin trigger IL-1β secretion by causing potassium efflux from which activates the NLRP3-ASC-caspase-1. We conclude that beyond their effects on fungal growth, these antifungal drugs directly activate the host's innate immunity.     

GNNQQNY—Investigation of Early Steps during Amyloid Formation

Protein aggregation has been implicated in the pathology of several neurodegenerative diseases, and a better understanding of how it proceeds is essential for the development of therapeutic strategies. Recently, the amyloidogenic heptapeptide GNNQQNY has emerged as a molecule of choice for fundamental studies of protein aggregation. A number of experimental and computational studies have examined the structure of the GNNQQNY aggregate. Less work, however, has been aimed at understanding its aggregation pathway. In this study, we present a detailed computational analysis of such a pathway. To that end, transition path sampling Monte Carlo simulations are used to examine the dimerization process. A statistical analysis of the reaction pathways shows that the dimerization reaction proceeds via a zipping mechanism, initiated with the formation of distinct contacts at the third residue (N). Asparagine residues are found to play a key role in the early stages of aggregation. And, contrary to previous belief, it is also shown that the tyrosine terminal group is not required to stabilize the dimer. In fact, an asparagine residue leads to faster aggregation of the peptide.     

Alterations in hemolymph and extrapallial fluid parameters in the Manila clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tapetis

In a recent study, we demonstrated the presence of defense factors, competent hemocytes and high enzymatic activities (peptidases, hydrolases, lytic, etc.), in the extrapallial fluid, located between the mantle and the shell, of the Manila clam, Ruditapes philippinarum. In Europe, this species is affected by brown ring disease, an epizootic disease caused by the bacterium Vibrio tapetis. The present work focused on the effect of the development of the disease on cellular and humoral defense parameters in the hemolymph and the extrapallial fluid of experimentally infected clams. Results indicate significant changes in total and dead hemocyte counts, as well as modifications in lysozyme activity and protein content, in the hemolymph and extrapallial fluid of challenged animals. Hemocyte counts and lysozyme activity increased significantly in the hemolymph, but particularly in the extrapallial fluid, where the highest values were observed. A healing (recalcification) process was observed 7 weeks following challenge, suggesting defense system efficiency at neutralizing the pathogen. These results are discussed with emphasis on the role of extrapallial fluids in the defense process against invading microorganisms.     

Characterization, dynamics, and ecological impacts of harmful Cochlodinium polykrikoides blooms on eastern Long Island, NY, USA

We report on the emergence of Cochlodinium polykrikoides blooms in the Peconic Estuary and Shinnecock Bay, NY, USA, during 2002–2006. Blooms occurred during late summer when temperatures and salinities ranged from 20 to 25 °C and 22 to 30 ppt, respectively. Bloom patches achieved cell densities exceeding 10⁵ ml⁻¹ and chlorophyll a levels exceeding 100 μg l⁻¹, while background bloom densities were typically 10³–10⁴ cells ml⁻¹. Light, scanning electron and ultrathin-section transmission electron microscopy suggested that cells isolated from blooms displayed characteristics of C. polykrikoides and provide the first clear documentation of the fine structure for this species. Sequencing of a hypervariable region of the large subunit rDNA confirmed this finding, displaying 100% similarity to other North American C. polykrikoides strains, but a lower similarity to strains from Southeast Asia (88–90%). Bioassay experiments demonstrated that 24 h exposure to bloom waters (>5 × 10⁴ cells ml⁻¹) killed 100% of multiple fish species (1-week-old Cyprinodon variegates, adult Fundulus majalis, adult Menidia menidia) and 80% of adult Fundulus heteroclitus. Microscopic evaluation of the gills of moribund fish revealed epithelial proliferation with focal areas of fusion of gill lamellae, suggesting impairment of gill function (e.g. respiration, nitrogen excretion, ion balance). Lower fish mortality was observed at intermediate C. polykrikoides densities (10³–10⁴ cells ml⁻¹), while fish survived for 48 h at cell densities below 1 × 10³ cells ml⁻¹. The inability of frozen and thawed-, or filtered (0.2 μm)-bloom water to cause fish mortality suggested that the thick polysaccharide layer associated with cell membranes and/or a toxin principle within this layer may be responsible for fish mortality. Juvenile bay scallops (Argopecten irradians) and American oysters (Crassostrea virginica) experienced elevated mortality compared to control treatments during a 9-day exposure to bloom water (5 × 10⁴ cells ml⁻¹). Surviving scallops exposed to bloom water also experienced significantly reduced growth rates. Moribund shellfish displayed hyperplasia, hemorrhaging, squamation, and apoptosis in gill and digestive tissues with gill inflammation specifically associated with areas containing C. polykrikoides cells. In summary, our results indicate C. polykrikoides blooms have become annual events on eastern Long Island and that bloom waters are capable of causing rapid mortality in multiple species of finfish and shellfish.     

Isolation of the pathogen Vibrio tapetis and defense parameters in brown ring diseased Manila clams Ruditapes philippinarum cultivated in England

The Manila clam Ruditapes philippinarum was introduced for aquacultural purposes to Europe in the 1970s. In 1987, brown ring disease (BRD), caused by Vibrio tapetis, appeared in clams cultivated in Brou?nou (Finistère, France) and later became increasingly widespread and was reported in cultivated and wild clams existing on the Atlantic coasts of France and Spain. The present study reports, for the first time, the presence of BRD in clams cultivated in England. The etiologic bacterium was isolated and identified using bacteriological and serological techniques. The defence response of affected clams was also studied and significant changes in the hematological and biochemical characteristics of hemolymph and extrapallial fluids were demonstrated. Significant mobilization of hemocytes toward the extrapallial fluids, in contact with the main site of infection (mantle-periostracal lamina area), was observed, suggesting a role for these pseudo-internal compartments in the preservation of clam health.     

The B lymphocyte adaptor molecule of 32 kilodaltons (Bam32) regulates B cell antigen receptor internalization

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is an adaptor that plays an indispensable role in BCR signaling. In this study, we found that upon BCR ligation, Bam32 is recruited to the plasma membrane where it associates with BCR complexes and redistributes and internalizes with BCRs. BCR ligation induced colocalization of Bam32 with lipid rafts, clathrin, and actin filaments. An inhibitor of Src family protein tyrosine kinases (PTKs) blocked both BCR-induced tyrosine phosphorylation of Bam32 and BCR internalization. Moreover, BCR internalization is impaired in Bam32-/- and Lyn-/- cells, and expression of Bam32 with a mutation of its tyrosine phosphorylation site (Y139F) inhibited BCR internalization. These data suggest that Bam32 functions downstream of Src family PTKs to regulate BCR internalization. Bam32 deficiency does not affect tyrosine phosphorylation of clathrin or the association of clathrin with lipid rafts upon BCR cross-linking. However, BCR-induced actin polymerization is impaired in Bam32-/- cells. Collectively, these findings indicate a novel role of Bam32 in connecting Src family PTKs to BCR internalization by an actin-dependent mechanism.     

Repetitive hit-and-run fluoroimmunoassay for T-2 toxin

A monoclonal antibody for T-2 toxin is converted to a Fab'-fluorescein derivative. The latter is specifically complexed onto a T-2 agarose gel. Fifteen successive doses of T-2 ranging from 1 to 50 ng are then repetitively and linearly detected using a column packed with a small volume (0.2 ml) of this gel without recharging with Fab'-fluorescein. For these assays the effluent from the column is monitored with a spectrofluorometer.