Postprandial metabolism and aversive response in rats fed a threonine-devoid diet

Lack of an indispensable amino acid in the diet induces a rapid reduction in food intake. In this study, we assessed whether the anorectic signal after ingestion of a meal lacking threonine originated from either direct perception of the decrease in plasma threonine or from an indirect effect related to increased postprandial amino acid catabolism and energy expenditure. We observed that 3 g of such a meal was sufficient to induce an aversive response to the diet within 2 h. Postprandial changes to plasma ammonia and urea, urinary urea, and energy metabolism did not differ from those measured after a control meal. In contrast, plasma threonine levels fell within 1 h after the meal. It is concluded that an increase in postprandial energy expenditure is not involved in the anorectic response to eating a threonine-devoid diet. The drop in plasma threonine levels may be a potential signal, but the fact that the decrease in food intake occurred 1 h after the decrease in plasma threonine questions a direct causal relationship.     

The first laminin G-type domain in the SHBG-like region of protein S contains residues essential for activation of the receptor tyrosine kinase sky

Vitamin K-dependent protein S and the product of growth-arrest-specific gene 6 (Gas6) both possess the ability to phosphorylate members of the Axl/Sky subfamily of receptor tyrosine kinases. However, Gas6 appears to be the bona fide ligand for these receptors in man, as human protein S has been demonstrated to activate murine Sky but not the human orthologue. In contrast, bovine protein S is able to stimulate human Sky despite its high degree of sequence identity with human protein S. The domain organisations of protein S and Gas6 are virtually identical and the C-terminal SHBG-like region, containing two globular (G) domains, has been shown to play a crucial role in the receptor stimulation. In order to further localise the area responsible for the interaction, a number of protein chimeras were used to stimulate human Sky. Each chimera had one part of the human protein S SHBG-like region replaced by the corresponding part of bovine protein S or human Gas6. We found that human protein S may indeed activate human Sky but only above physiological plasma concentrations. The human-bovine protein S chimeras provided new information implying that the first G domain contains critical residues for the interaction with the Sky receptor. Moreover, these residues do not seem to be clustered but rather to be distributed at various positions in the first G domain.     

Novel approach to nucleoside-5'-(1-hydroxymethylene-1, 1-bisphosphonates): synthesis of new AZT analogues

An efficient synthetic method of nucleoside-5'-(1-hydroxymethylene-1,1-bisphosphonates) is reported here. The procedure was optimized with 3'-protected thymidine and then applied to synthesis of new AZT analogues.     

Purification and characterization of Atlantic salmon (Salmo salar) fibrinogen

This study describes a purification protocol of salmon fibrinogen that gives a consumable and highly clottable fibrinogen. Some characteristics of salmon and human fibrinogen are compared. Fibrinogen was purified from barium sulphate adsorbed plasma of Atlantic salmon, using two steps of 25% ammonium sulphate precipitation followed by ultrafiltration. The clottability of the purified salmon fibrinogen was 91%. The Aalpha chains of salmon fibrinogen were heterogeneous with a molecular mass of 90-110 kDa, compared to approximately 67 kDa of human fibrinogen Aalpha chains. The Bbeta and gamma chains of salmon and human fibrinogen had molecular masses of approximately 55 and 50 kDa, respectively. Western blotting revealed that polyclonal rabbit anti-human fibrinogen antibodies had affinity for the gamma chains of salmon fibrinogen, making it possible to study factor XIII activity in purified salmon fibrinogen. Cross-linking of either gamma-gamma or gamma-alpha chains was not detected upon incubation of the purified fibrinogen with thrombin and calcium alone, but was detected when clotting was performed in plasma indicating absence of factor XIII activity in the purified product.     

Specific fluorescent tracers. Imaging and applications for photodynamic therapy

Our main objective is to enlarge the fluorescence use in biosciences, with especially the photodynamic therapy (PDT) used for cancer treatment as one of the target applications. Meta-tetra(hydroxyphenyl)chlorin (m-THPC) is a second-generation photosensitiser, applied in photodynamic therapy. The localisation of this sensitiser as well as its induced cell death mechanisms in human breast cancer cells (MCF-7 and its resistant subline MCF-7DXR, DXR: doxorubicin) were evaluated using fluorescence microscopy. In addition, we will present two additional routes, whose aims are to create new features to respond to the PDT questioning: firstly, the synthesis of fluorescent tracers, with a particular attention to the presence of hydrophilic groups (glucosamine ring) on the basic fluorophore structure to orientate the localisation of the probe and, secondly, the use of scanning near-field optical microscopy to reach a better resolution for the fluorescence microscopy analysis.     

Characterization of the EGF-like module pair 3-4 from vitamin K-dependent protein S using NMR spectroscopy reveals dynamics on three separate time scales and extensive effects from calcium binding

Protein S, a cofactor of anticoagulant activated protein C, exhibits three high-affinity Ca(2+)-binding sites in a region comprising four EGF modules. The EGF 3-4 module pair constitutes the smallest fragment that retains one high-affinity Ca(2+)-binding site and is therefore useful for investigation of the structural basis of the unusually high-affinity Ca(2+) binding compared to other EGF-containing proteins characterized so far. Extensive chemical shift effects caused by Ca(2+) binding to the EGF 3-4 module pair are observed, particularly from Ca(2+) binding to the high-affinity site in EGF 4. Ca(2+) binding to the high-affinity site in EGF 4 and the low-affinity site in EGF 3 is associated with slow and fast exchange on the NMR time-scale, respectively. We show the presence of two isoforms, characterized by a cis or trans Lys 167-Pro 168 peptide bond, that do not convert on time scales that were accessible to the experiments (k(ex) < 0.2 s(-1)). Both conformers have similar Ca(2+) affinities and backbone dynamics. Further, broadening of (1)H resonances involving residues in the major beta-sheet of EGF 3 and (15)N exchange terms, primarily in the N-terminal part of the protein, indicate the presence of slow exchange on a microsecond to millisecond time scale. (15)N spin relaxation data suggest that the module pair has a well-defined relative orientation between EGF modules 3 and 4 and has a significantly anisotropic rotational diffusion tensor in solution.     

Light chain germ-line genes and the immune response to 2-phenyloxazolone.

Direct sequencing of mRNA has shown that the early primary response of the BALB/c mouse to the hapten 2-phenyloxazolone is dominated by antibodies with a particular light chain, V kappa-Ox1. Although the V kappa-Ox1 sequence is still commonly expressed later in the response it now includes a number of nucleotide changes. From two independent BALB/c germ-line DNA libraries 13 different genes hybridizing to a V kappa-Ox1 probe were isolated and characterized. Two are identical to mRNA sequences found in the early primary response, one of which is the V kappa-Ox1 sequence. None of the germ-line clones show the characteristic nucleotide changes contained in the late anti-phenyloxazolone light chain mRNAs. These results demonstrate that the V kappa-Ox1 sequence used in the early primary response is entirely encoded by the germ-line and further substantiate the importance of somatic mutations in the maturation of the anti-phenyloxazolone response. The statistical analysis of the data shows that the V kappa-Ox1 related germ-line gene family contains greater than 20 and probably less than 50 genes.     

Seasonal Dynamics of Atlantic Herring (Clupea harengus L.) Populations Spawning in the Vicinity of Marginal Habitats

Gillnet sampling and analyses of otolith shape, vertebral count and growth indicated the presence of three putative Atlantic herring (Clupea harengus L.) populations mixing together over the spawning season February–June inside and outside an inland brackish water lake (Landvikvannet) in southern Norway. Peak spawning of oceanic Norwegian spring spawners and coastal Skagerrak spring spawners occurred in March–April with small proportions of spawners entering the lake. In comparison, spawning of Landvik herring peaked in May–June with high proportions found inside the lake, which could be explained by local adaptations to the environmental conditions and seasonal changes of this marginal habitat. The 1.85 km² lake was characterized by oxygen depletion occurring between 2.5 and 5 m depth between March and June. This was followed by changes in salinity from 1–7‰ in the 0–1 m surface layer to levels of 20–25‰ deeper than 10 m. In comparison, outside the 3 km long narrow channel connecting the lake with the neighboring fjord, no anoxic conditions were found. Here salinity in the surface layer increased over the season from 10 to 25‰, whereas deeper than 5 m it was stable at around 35‰. Temperature at 0–5 m depth increased significantly over the season in both habitats, from 7 to 14°C outside and 5 to 17°C inside the lake. Despite differences in peak spawning and utilization of the lake habitat between the three putative populations, there was an apparent temporal and spatial overlap in spawning stages suggesting potential interbreeding in accordance with the metapopulation concept.