Viability assessed with the most probable number dilution culture method after chemical treatment of ballast water reveals the presence of false negatives from an approved vital stain method.

The present study compares the CMFDA/FDA + motility- and the Most Probable Number (MPN) Dilution Culture + Motility methods for testing the viability of ≥10-<50 µm organisms in chlorine treated ballast water. The results of both methods were within the regulatory compliance criterion <10 organisms/mL, but the MPN-method revealed that growth-outs did occur. While the CMFDA/FDA method showed <0.5 organisms/mL, the MPN-method gave approx. 6 organisms/mL. This demonstrated that false negatives, i.e. living but not stained organisms, may occur when using the CMFDA/FDA-method for compliance testing of chemical treated ballast water. Organisms surviving the treatment were primarily the dinoflagellate Scrippsiella sp. and various coccoid chlorophytes present in a brackish- and freshwater test, respectively. It is suggested that their resilience to the chemical treatment is the ability to transform into a temporary cyst (Scrippsiella sp.) or the presence of a chemical resistant cell wall (certain chlorophytes).

Inherent lack of CMFDA/FDA staining in certain algae and its implication for ballast water testing.

The approved method for testing the efficacy of ballast water management systems with respect to killing 10-50 µm organisms uses movements of the organisms or the vital stains CMFDA/FDA. The present study demonstrates that certain freshwater coccoid chlorophytes, known or suspected to contain a highly resistant cell wall component (algaenan), stain poorly with CMFDA/FDA, resulting in false negatives. The staining rates for the most dominant species were determined and were approx. 3-70 %. The use of Crystal Violet as an indicator for the presence of algaenan gave inconclusive results. The number of the 10-50 µm organisms in a small pond was found to be 10,183 organisms/mL (Lugol's fixed sample) vs. 2335 organisms/mL (CMFDA/FDA-stained sample). Using the staining rates obtained, it was estimated that the number of false negatives could make 40-50 %. The implications for biological performance evaluation of ballast water management systems are discussed.

Haslea silbo, A Novel Cosmopolitan Species of Blue Diatoms.

Specimens of a new species of blue diatoms from the genus Haslea Simonsen were discovered in geographically distant sampling sites, first in the Canary Archipelago, then North Carolina, Gulf of Naples, the Croatian South Adriatic Sea, and Turkish coast of the Eastern Mediterranean Sea. An exhaustive characterization of these specimens, using a combined morphological and genomic approach led to the conclusion that they belong to a single new to science cosmopolitan species, Haslea silbo sp. nov. A preliminary characterization of its blue pigment shows similarities to marennine produced by Haslea ostrearia, as evidenced by UV-visible spectrophotometry and Raman spectrometry. Life cycle stages including auxosporulation were also observed, providing data on the cardinal points of this species. For the two most geographically distant populations (North Carolina and East Mediterranean), complete mitochondrial and plastid genomes were sequenced. The mitogenomes of both strains share a rare atp6 pseudogene, but the number, nature, and positions of the group II introns inside its cox1 gene differ between the two populations. There are also two pairs of genes fused in single ORFs. The plastid genomes are characterized by large regions of recombination with plasmid DNA, which are in both cases located between the ycf35 and psbA genes, but whose content differs between the strains. The two sequenced strains hosts three plasmids coding for putative serine recombinase protein whose sequences are compared, and four out of six of these plasmids were highly conserved.

Short-term tissue permeability actions of dextran sulfate sodium studied in a colon organ culture system.

Dextran sulfate sodium (DSS)-induced colitis is the most commonly used animal model for inflammatory bowel diseases. However, the precise molecular action of DSS, in particular its initial effect on the epithelial tissue permeability, is still poorly understood. In the present work, organ culture of mouse - and pig colon explants were performed for 1-2 h in the presence/absence of 2% DSS together with polar- and lipophilic fluorescent probes. Probe permeability was subsequently assessed by fluorescence microscopy. DSS rapidly increased paracellular permeability of 70-kDa dextran without otherwise affecting the overall epithelial integrity. FITC-conjugated DSS likewise permeated the epithelial barrier and strongly accumulated in nuclei of cells scattered in the lamina propria. By immunolabeling, plasma cells, T cells, macrophages, mast cells, and fibroblasts were identified as possible targets for DSS, indicating that accumulation of the polyanion in nuclei was not confined to a particular type of cell in the lamina propria. In contrast, colonocytes were rarely targeted by DSS, but as visualized by transmission electron microscopy, it induced the formation of vacuole-like structures in the intercellular space between adjacent epithelial cells. Nuclei of various cell types in the lamina propria, including both cells of the innate and adaptive immune system, are novel targets for a rapid action of DSS, and from previous in vitro studies, polyanions like DSS are known to disrupt nucleosomes by binding to the histones. We therefore propose that nuclear targeting is one way whereby DSS exerts its inflammatory action as a colitogen in animal models of inflammatory bowel diseases.

Probing paracellular -versus transcellular tissue barrier permeability using a gut mucosal explant culture system.

Intestinal permeation enhancers (PEs), i.e. agents improving oral delivery of therapeutic drugs with poor bioavailability, may typically act by two principally different mechanisms: to increase either transcellular -or paracellular passage across the epithelium. With the aim to define these different modes of action in a small intestinal mucosal explant system, the transcellular-acting PE sodium dodecyl sulfate (SDS) was compared to the paracellular-acting PE ethylenediaminetetraacetic acid (EDTA), using several fluorescent polar - and lipophilic probes. Here, SDS rendered the enterocyte cell membranes leaky for the relatively small polar tracers Lucifer yellow and a 3 kD Texas red-conjugated dextran, but most conspicuously SDS blocked constitutive endocytosis from the brush border. In contrast, the main action of EDTA was to increase paracellular passage across the epithelium of both polar probes, including 10 - and 70 kDa dextrans and lipophilic probes, visualized by distinct stripy lateral staining of enterocytes and/or accumulation in the lamina propria. In addition, EDTA caused a loss of epithelial cell polarity by opening tight junctions for diffusion of domain-specific basolateral/apical cell membrane protein markers into the opposite domains. By transmission electron microscopy, SDS caused the formation of vacuoles and vesicle-like structures at the lateral cell membranes. In contrast, EDTA led to a bulging of the whole enterocyte apex, resulting in a "cobblestone" appearance of the epithelium, probably caused by an extreme contraction of the perijunctional actomyosin ring. We conclude that the mucosal explant system is a convenient model for predicting transcellular/paracellular modes of action of novel prospective PEs.

Probing the Action of Permeation Enhancers Sodium Cholate and N-dodecyl-ß-D-maltoside in a Porcine Jejunal Mucosal Explant System.

The small intestinal epithelium constitutes a major permeability barrier for the oral administration of therapeutic drugs with poor bioavailability, and permeation enhancers (PEs) are required to increase the paracellular and/or transcellular uptake of such drugs. Many PEs act as surfactants by perturbing cell membrane integrity and causing permeabilization by leakage or endocytosis. The aim of the present work was to study the action of sodium cholate (NaC) and N-dodecyl-ß-D-maltoside (DDM), using a small intestinal mucosal explant system. At 2 mM, both NaC and DDM caused leakage into the enterocyte cytosol of the fluorescent probe Lucifer Yellow, but they also blocked the constitutive endocytotic pathway from the brush border. In addition, an increased paracellular passage of 3-kDa Texas Red Dextran into the lamina propria was observed. By electron microscopy, both PEs disrupted the hexagonal organization of microvilli of the brush border and led to the apical extrusion of vesicle-like and amorphous cell debris to the lumen. In conclusion, NaC and DDM acted in a multimodal way to increase the permeability of the jejunal epithelium both by paracellular and transcellular mechanisms. However, endocytosis, commonly thought to be an uptake mechanism that may be stimulated by PEs, was not involved in the transcellular process.

Impact of cell-penetrating peptides (CPPs) melittin and Hiv-1 Tat on the enterocyte brush border using a mucosal explant system.

"Cell penetrating peptides" (CPPs) are natural or synthetic peptides with the ability to interact with cell membranes in order to enter cells and/or deliver cargo. They attract considerable interest as permeation enhancers for oral delivery of therapeutic drugs with poor bioavailability, such as proteins or DNA. A main barrier is the intestinal epithelium where passage needs to proceed through a paracellular -and/or a transcellular pathway. Using an organ cultured mucosal explant model system and a selection of fluorescent polar -and lipophilic tracers, the aim of the present study was to investigate the interaction of two CPPs, melittin and Hiv-1 Tat, with the enterocyte brush border. Melittin belongs to the amphipathic class of CPPs, and within 0.5-1 h it bound to, and penetrated, the enterocyte brush border, causing leakage into the cytosol and increased paracellular passage into the lamina propria. Surprisingly, melittin also abolished endocytosis of tracers from the brush border into early endosomes in the terminal web region (TWEEs), excluding any permeation enhancing effect via such an uptake mechanism. Electron microscopy revealed that melittin caused an elongation of the brush border microvilli and a reduction in their diameter. HIV-1 Tat is a cationic CPP that is internalized by cells due to a sequence, mainly of arginines, from residue 49 to 57, and a peptide containing this sequence permeabilized enterocytes to a polar tracer by a leakage into the cytosol. In conclusion, the CPPs studied acted by causing leakage of tracers into the enterocyte cytosol, not by inducing endocytosis.

The Rainwater Rock-pool Dinoflagellate Nottbeckia ochracea gen. et comb. nov. (syn.: Hemidinium ochraceum) - A Fine-structural and Molecular Study with Emphasis on the Motile Stage.

The rarely recorded dinoflagellate Hemidinium ochraceum Levander was isolated from a rainwater rock-pool in Finland and the motile stage studied by light- and electron microscopy and molecular sequencing. The ultrastructure of the motile stage revealed several features not previously observed in other dinoflagellates, e.g. thecal plates with inwards 'knobs' and a peculiar corrugated left sulcal plate. The apparent lack of the R2 and R4 flagellar roots was also unusual. The typical plate tabulation was 4', 2a, 8″, cx, 4c,?s, 3‴, cy, 1p, 2″″, cx and cy being extra plates associated with the cingulum, but intraspecific variations were observed in shape and number of plates. Phylogenetic analyses of nuclear-encoded LSU rDNA and concatenation of LSU and SSU rDNA sequences showed that H. ochraceum was only distantly related to Hemidinium nasutum (the type species of Hemidinium). The immotile coccoid stage resembled Gloeodinium montanum and a weakly supported relationship between the two was only inferred in the analyses based on LSU rDNA. Hemidinium ochraceum showed some resemblance to Rufusiella insignis sensu Richards, but the true identity of this species and of genus Rufusiella is uncertain. A new genus, Nottbeckia, is erected to accommodate H. ochraceum, as Nottbeckia ochracea gen. et comb. nov.

Intestinal surfactant permeation enhancers and their interaction with enterocyte cell membranes in a mucosal explant system.

Intestinal permeation enhancers (PEs) are agents aimed to improve oral delivery of therapeutic drugs with poor bioavailability. The main permeability barrier for oral delivery is the intestinal epithelium, and PEs act to increase the paracellular and/or transcellular passage of drugs. Transcellular passage can be achieved by cell membrane permeabilization and/or by endocytic uptake and subsequent transcytosis. One broad class of PEs is surfactants which act by inserting into the cell membrane, thereby perturbing its integrity, but little is known about how the dynamics of the membrane are affected. In the present work, the interaction of the surfactants lauroyl-L-carnitine, 1-decanoyl-rac-glycerol, and nonaethylene glycol monododecyl ether with the intestinal epithelium was studied in organ cultured pig jejunal mucosal explants. As expected, at 2 mM, these agents rapidly permeabilized the enterocytes for the fluorescent polar tracer lucifer yellow, but surprisingly, they all also blocked both constitutive -and receptor-mediated pathways of endocytosis from the brush border, indicating a complete arrest of apical membrane trafficking. At the ultrastructural level, the PEs caused longitudinal fusion of brush border microvilli. Such a membrane fusogenic activity could also explain the observed formation of vesicle-like structures and large vacuoles along the lateral cell membranes of the enterocytes induced by the PEs. We conclude that the surfactant action of the PEs selected in this study not only permeabilized the enterocytes, but profoundly changed the dynamic properties of their constituent cell membranes.

IgG trafficking in the adult pig small intestine: one- or bidirectional transfer across the enterocyte brush border?

Immunoglobulin G (IgG) transfer in opposite directions across the small intestinal brush border serves different purposes in early life and in adulthood. In the neonate, maternal IgG is taken up from the gut lumen into the blood, conferring passive immunity to the offspring, whereas in the adult immunoglobulins, including IgG made by plasma cells in the lamina propria, are secreted via the brush border to the lumen as part of the mucosal defense. Here, IgG has been proposed to perform a luminal immune surveillance which eventually includes a reuptake through the brush border as pathogen-containing immune complexes. In the present work, we studied luminal uptake of FITC-conjugated and gold-conjugated IgG in cultured pig jejunal mucosal explants. After 1 h, binding to the brush border was seen in upper crypts and lower parts of the villi. However, no endocytotic uptake into EEA-1-positive compartments was detected, neither at neutral nor acidic pH, despite an ongoing constitutive endocytosis from the brush border, visualized by the polar tracer CF594. The 40-kDa neonatal Fc receptor, FcRn, was present in the microvillus fraction, but noteworthy, a 37 kDa band, most likely a proteolytic cleavage product, bound IgG in a pH-dependent manner more efficiently than did the full-length FcRn. In conclusion, our work does not support the theory that bidirectional transfer of IgG across the intestinal brush border is part of the luminal immune surveillance in the adult.

Comparative Analysis of Membrane Lipids in Psychrophilic and Mesophilic Freshwater Dinoflagellates.

Here we report the lipid profiles of ten dinoflagellate species originating from different freshwater habitats and grown at 4, 13, or 20°C akin to their natural occurrence. Lipids were determined by High Performance Liquid Chromatography-ElectroSpray Ionization-Mass Spectrometry in positive and negative ion modes. Besides the well-studied monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) lipids, our study revealed the presence of intact molecular lipid species of trigalactosyldiacylglycerols, betaine diacylglyceryl-carboxyhydroxymethylcholine, sulfolipid sulfoquinovosyldiacylglycerols (SQDG) and phospholipids, in particular phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. In multivariate ordination, the freshwater dinoflagellates studied could be distinguished into two groups based on their lipid profiles. Peridinium aciculiferum, Borghiella dodgei, B. tenuissima and Tovellia coronata belonged to group 1 while Ceratium cornutum, Gymnodinium palustre, Jadwigia applanata, P. cinctum, P. willei, and P. gatunense belonged to group 2. Indicator species analysis evidenced that group 1 was characterized by 36:9 MGDG and 36:9 DGDG and group 2 by 38:9 and 38:10 MGDG, 38:9 and 38:10 DGDG and 34:1 SQDG. We suggest that the grouping of dinoflagellates indicated their range of temperature tolerance. Furthermore, non-thylakoid lipids were linked to dinoflagellate phylogeny based on the large ribosomal sub-unit (28S LSU) rather than their temperature tolerance. Thus certain lipids better reflected habitat adaptation while other lipids better reflected genetic diversity.

C4.4A gene ablation is compatible with normal epidermal development and causes modest overt phenotypes.

C4.4A is a modular glycolipid-anchored Ly6/uPAR/α-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.

Small molecule pinocytosis and clathrin-dependent endocytosis at the intestinal brush border: Two separate pathways into the enterocyte.

Pinocytosis at the small intestinal brush border was studied in postweaned porcine cultured mucosal explants, using the fluorescent polar probes Alexa hydrazide (AH, MW 570), Texas red dextran (TRD, MW ~ 3000), and Cascade blue dextran (CBD, MW ~ 10,000). Within 1 h, AH appeared in a string of subapical punctae in enterocytes, indicative of an ongoing constitutive pinocytosis. By comparison, TRD was taken up less efficiently into the same compartment, and no intracellular labeling of CBD was detectable, indicating that only small molecules are pinocytosed from the postweaned gut lumen. AH remained in the terminal web region in EEA-1-positive endosomes ("TWEEs") for at least 2 h, implying that the pinocytic uptake does not proceed towards a transcytic pathway. Like AH, cholera toxin B subunit (CTB) was readily internalized, but the two probes appeared in completely non-overlapping subapical compartments, indicating the existence of two different uptake mechanisms operating simultaneously at the brush border. CTB is internalized by clathrin-dependent receptor mediated endocytosis, but surprisingly the toxin also caused a rapid disappearance from the apical cell surface of two major brush border enzymes, alkaline phosphatase and aminopeptidase N, demonstrating the disruptive effect of this pathway. By immunofluorescence, caveolin-1 was hardly detectable in enterocytes, arguing against a caveolae-mediated uptake of AH, whereas the pinocytosis/phagocytosis inhibitors dimethyl amiloride and cytochalasin D both arrested AH uptake. We propose that the constitutive pinocytic mechanism visualized by AH contributes to maintenance of membrane homeostasis and to enrich the contents of lipid raft constituents at the brush border.

Organ Culture as a Model System for Studies on Enterotoxin Interactions with the Intestinal Epithelium.

Studies on bacterial enterotoxin-epithelium interactions require model systems capable of mimicking the events occurring at the molecular and cellular levels during intoxication. In this chapter, we describe organ culture as an often neglected alternative to whole-animal experiments or enterocyte-like cell lines. Like cell culture, organ culture is versatile and suitable for studying rapidly occurring events, such as enterotoxin binding and uptake. In addition, it is advantageous in offering an epithelium with more authentic permeability/barrier properties than any cell line, as well as a subepithelial lamina propria, harboring the immune cells of the gut mucosa.

A longitudinal study of coordination in mother-infant vocal interaction from age 4 to 10 months.

This is a longitudinal study of development in coordinated mother-infant vocal interaction from 4 to 10 months (N = 41) focusing on the development of turn-taking patterns and time spent in coordinated vocal interaction. Data analyses were carried out using multistate analysis. Both mothers and infants were found to coordinate their own vocalizations with their partners' vocalizations. The infants had a predominant pattern of covocalizing, whereas the mothers had a predominant turn-taking pattern at all ages (4, 7, and 10 months). However, a significant reduction in the duration of covocalization was found between 4 and 7 months, due to less covocalizing compared to turn-taking from the infants. In addition, time spent in coordinated vocal interaction increased significantly between 4 and 7 months and a development for the infants was found from repeated vocalizations toward single vocalizations between maternal turns. Taken together, these findings indicate a developmental process of fine-tuning and increasing the turn-taking format for vocal communication, characterizing adult communication, before the development of speech. In addition, our results indicate that this development starts earlier and is more prolonged than has been hypothesized from previous research. (PsycINFO Database Record

Recent radiation in a marine and freshwater dinoflagellate species flock.

Processes of rapid radiation among unicellular eukaryotes are much less studied than among multicellular organisms. We have investigated a lineage of cold-water microeukaryotes (protists) that appear to have diverged recently. This lineage stands in stark contrast to known examples of phylogenetically closely related protists, in which genetic difference is typically larger than morphological differences. We found that the group not only consists of the marine-brackish dinoflagellate species Scrippsiella hangoei and the freshwater species Peridinium aciculiferum as discovered previously but also of a whole species flock. The additional species include Peridinium euryceps and Peridinium baicalense, which are restricted to a few lakes, in particular to the ancient Lake Baikal, Russia, and freshwater S. hangoei from Lake Baikal. These species are characterized by relatively large conspicuous morphological differences, which have given rise to the different species descriptions. However, our scanning electron microscopic studies indicate that they belong to a single genus according to traditional morphological characterization of dinoflagellates (thecal plate patterns). Moreover, we found that they have identical SSU (small subunit) rDNA fragments and distinct but very small differences in the DNA markers LSU (large subunit) rDNA, ITS2 (internal transcribed spacer 2) and COB (cytochrome b) gene, which are used to delineate dinoflagellates species. As some of the species co-occur, and all four have small but species-specific sequence differences, we suggest that these taxa are not a case of phenotypic plasticity but originated via recent adaptive radiation. We propose that this is the first clear example among free-living microeukaryotes of recent rapid diversification into several species followed by dispersion to environments with different ecological conditions.

The subcellular localization of GABA transporters and its implication for seizure management.

The ability to modulate the synaptic GABA levels has been demonstrated by using the clinically effective and selective GAT1 inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid]. N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) which not only inhibits GAT1 like tiagabine but also BGT1 has been shown to modulate extrasynaptic GABA levels. The simultaneous inhibition of synaptic and extrasynaptic GABA transporters using tiagabine and EF1502, respectively has been demonstrated to exert a synergistic anticonvulsant effect in several seizure models in mice. The pharmacological profile of these and similar compounds has been thoroughly investigated in in vitro systems, comparing the GAT subtype selectivity with the ability to inhibit GABA uptake in primary cultures of neurons and astrocytes. However, an exact explanation has not yet been found. In the present study, the ability of GATs to form homo and/or heterodimers was investigated as well as to which membrane micro environment the GATs reside. To investigate dimerization of GATs, fusion proteins of GATs tagged with either yellow fluorescent protein or cerulean fluorescent protein were made and fluorescence resonance energy transfer (FRET) was measured. It was found that GATs form both homo- and hetero-dimers in N2A and HEK-293 cells. Microdomain localization of GATs as investigated by detergent resistant membrane fractions after treatment of tissue with Brij-98 or Triton X-100 revealed that BGT1 and GAT1 mostly localize to non-membrane rafts independent of the detergent used. However, GAT3 localizes to membrane rafts when using Brij-98. Taken together, these results suggest that the observed hetero dimerization of GATs in the FRET study is unlikely to have functional implications since the GATs are located to very different cellular compartments and cell types.

Okadaic acid: a rapid inducer of lamellar bodies in small intestinal enterocytes.

Okadaic acid (OA) is a polyether fatty acid produced by marine dinoflagellates and the causative agent of diarrhetic shellfish poisoning. The effect of OA on apical endocytosis in the small intestine was studied in organ cultured porcine mucosal explants. Within 0.5-1 h of culture, the toxin caused hyper protein phosphorylation, but no detectable loss of cell polarity or cytoskeletal integrity of the enterocytes. Using a fluorescent membrane marker, FM dye, endocytosis from the brush border was affected by the toxin. Although constitutive uptake into subapical terminal web-localized early endosomes (TWEEs) occurred unimpeded in the presence of OA, FM condensed in larger subapical structures by 1 h, implying a perturbed endosomal trafficking/maturation. The fluorescent lysosomotropic agent Lysotracker revealed induction of large lysosomal structures by OA. Endocytosis from the brush border was studied at the electron microscopic level using the membrane-impermeable marker Ruthenium Red (RR). Like FM dye, RR was taken up into TWEEs and multivesicular bodies (MVBs). However, OA induced the formation of a large number of lamellar bodies (LBs), a type of lysosome-related organelles. LBs are the hallmark of phospholipidosis, a pathological condition characterized by lysosomal phospholipid accumulation. Phospholipidosis is observed in acquired lysosomal storage diseases and is induced by a large number of cationic amphiphilic drugs. Unlike the latter, however, OA does not act by accumulating in acidic organelles, implying a different toxic mechanism of action. We propose that rapid induction of LBs, an indicator of phospholipidosis, should be included in the future toxicity profile of OA.

Marennine, promising blue pigments from a widespread Haslea diatom species complex.

In diatoms, the main photosynthetic pigments are chlorophylls a and c, fucoxanthin, diadinoxanthin and diatoxanthin. The marine pennate diatom Haslea ostrearia has long been known for producing, in addition to these generic pigments, a water-soluble blue pigment, marennine. This pigment, responsible for the greening of oysters in western France, presents different biological activities: allelopathic, antioxidant, antibacterial, antiviral, and growth-inhibiting. A method to extract and purify marennine has been developed, but its chemical structure could hitherto not be resolved. For decades, H. ostrearia was the only organism known to produce marennine, and can be found worldwide. Our knowledge about H. ostrearia-like diatom biodiversity has recently been extended with the discovery of several new species of blue diatoms, the recently described H. karadagensis, H. silbo sp. inedit. and H. provincialis sp. inedit. These blue diatoms produce different marennine-like pigments, which belong to the same chemical family and present similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms thus present a commercial potential for aquaculture, cosmetics, food and health industries.

Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane.

The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB) was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.