Pelagic larvae of New England intertidal gastropods II. Anachis avara

Summary The egg case, pelagic larvae, and young juvenile of Anachis avara Say, a species of prosobranch gastropod belonging to the family Columbellidae, are described and figured from specimes reared in the laboratory.

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Zusammenfassung Die Arbeit beschreibt die Eierkapseln, die pelagischen Larven und die Jugendformen von Anachis avara Say, einer prosobranchen Gastropoden-Spezies, die zur Familie der Columbelliden gehört. Abbildungen von Exemplaren, die im Laboratorium aufgezogen wurden, werden wiedergegeben.

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Contribution number 1341, Woods Hole Oceanographic Institution. This research was supported by N.S.F. grant no. 17883.     

Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.     

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.     

A comparison of national approaches to setting ecological status boundaries in phytobenthos assessment for the European Water Framework Directive: results of an intercalibration exercise

The European Union (EU)’s Water Framework Directive (WFD) requires that all Member States participate in intercalibration exercises in order to ensure that ecological status concepts and assessment levels are consistent across the EU. This paper describes one such exercise, performed by the countries in the Central/Baltic Geographical Intercalibration Group stretching from Ireland in the west to Estonia in the east and from the southern parts of Scandinavia to the northern regions of Spain and Italy (but excluding alpine regions, which were intercalibrated separately). In this exercise, methods used to measure ecological status of rivers using benthic diatoms were compared. Ecological status is estimated as the ratio between the observed value of a biological element and the value expected in the absence of significant human impact. Approaches to defining the ‘reference sites’, from which these ‘expected’ values were derived, varied from country to country. Minimum criteria were established as part of the exercise but there was still considerable variation between national reference values, reflecting typological differences that could not be resolved during the exercise. A simple multimetric index was developed to compare boundary values using two widely used diatom metrics. Boundary values for high/good status and good/moderate status set by each participant were converted to their equivalent values of this intercalibration metric using linear regression. Variation of ±0.05 EQR units around the median value was considered to be acceptable and the exercise provided a means for those Member States who fell significantly above or below this line to review their approaches and, if necessary, adjust their boundaries. Handling editor: J. Padisak     

Supercooling ability in two populations of the land snail Helix pomatia (Gastropoda: Helicidae) and ice-nucleating activity of gut bacteria

The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gotaland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T=5 degrees C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T(c)=-6.4+/-0.8 degrees C), significantly greater, however, than the supercooling capacity of the population from France (T(c)=-4.6+/-1.4 degrees C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T=20 degrees C), both populations exhibited a similar high T(c) (-2.0+/-1.0 degrees C). The lower T(c) of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg(-1)) and active (165-215 mOsm kg(-1)) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than -9 degrees C. Only the strain Kluyvera sp. initiated nucleation at -9 degrees C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of hibernating snail populations, triggering changes in osmolyte concentration, rather than the presence of endogenous ice-nucleating-active bacteria, accounts for fluctuations in their T(c).     

Evaluation of HepaRG cells for the assessment of indirect drug-induced hepatotoxicity using INH as a model substance

HepaRG cells are widely used as an in vitro model to assess drug-induced hepatotoxicity. However, only few studies exist so far regarding their suitability to detect the effects of drugs requiring a preceding activation via the cytochrome P450 (CYP) system. A prototypic substance is the anti-tuberculosis agent INH, which is metabolized into N-acetylhydrazine, which then triggers hepatotoxicity. Therefore, the aim of the present study was to test if this effect can also be detected in HepaRG cells and if it can be counteracted by the known hepatoprotectant silibinin. For this purpose, differentiated HepaRG cells were treated with increasing concentrations of INH (0.1–100 mM) or 10 mM INH plus escalating concentrations of silibinin (1–100 µM). After 48 h of treatment, cell morphology and parameters indicating cell vitality, oxidative stress, and liver cell function were assessed. High concentrations of INH led to severe histopathological changes, reduced cell vitality and glutathione content, increased LDH and ASAT release into the medium, enhanced lipid peroxidation, and elevated cleaved caspase-3 expression. Additionally, glycogen depletion and reduced biotransformation capacity were seen at high INH concentrations, whereas at low concentrations an induction of biotransformation enzymes was noticed. Silibinin caused clear-cut protective effects, but with few parameters INH toxicity was even aggravated, most probably due to increased metabolization of INH into its toxic metabolite. In conclusion, HepaRG cells are excellently suited to evaluate the effects of substances requiring prior toxification via the CYP system, such as INH. They additionally enable the identification of complex substance interactions.