Improved isolation procedure for azaspiracids from shellfish, structural elucidation of azaspiracid-6, and stability studies

Azaspiracids are a group of lipophilic polyether toxins produced by the small dinoflagellate Azadinium spinosum. They may accumulate in shellfish and can result in illnesses when consumed by humans. Research into analytical methods, chemistry, metabolism, and toxicology of azaspiracids has been severely constrained by the scarcity of high-purity azaspiracids. Consequently, since their discovery in 1995, considerable efforts have been made to develop methods for the isolation of azaspiracids in sufficient amounts and purities for toxicological studies, in addition to the preparation of standard reference materials. A seven-step procedure was improved for the isolation of azaspiracids-1-3 (1, 2, and 3) increasing recoveries 2-fold as compared to previous methods and leading to isolation of sufficiently purified azaspiracid-6 (6) for structural determination by NMR spectroscopy. The procedure, which involved a series of partitioning and column chromatography steps, was performed on 500 g of Mytilus edulis hepatopancreas tissue containing ~14 mg of 1. Overall yields of 1 (52%), 2 (43%), 3 (43%), and 6 (38%) were good, and purities were confirmed by NMR spectroscopy. The structure of 6 was determined by one- and two-dimensional NMR spectroscopy and mass spectrometry. The stability of 6 relative to 1 was also assessed in three solvents in a short-term study that demonstrated the greatest stability in aqueous acetonitrile.     

High-efficiency regnerationin vitro from potato petioles with intact leaflets

The shoot/plantlet regenerationin vitro of seven potato (Solanum tuberosum L.) cultivars from petioles with intact leaflets was assessed using six treatment combinations-a basal medium with or without silver thiosul-phate (STS) or thidiazuron (TDZ) at two concentrations (2 or 0.5 mg/l) of the indoleacetic acid (IAA). The basal medium consisted of Murashige and Skoog (MS) salts and vitamins supplemented with 3 mg/1 6-benzylaminopurine, and 1 mg/1 gibberellic acid, 30 g/l sucrose, and 7.0 g/l PHYTOAGAR. Two full sets repeats and one partial set repeat of independent experiments were conducted and all produced similar results. Silver thiosulphate decreased the regeneration frequency and number of shoots per callus. No significant changes were observed with thidiazuron. Regeneration rates of (100% ) with up to 20 shoots/plantlets per callus were achieved at 2 mg/1 IAA with cultivars Désirée, Kennebec, Niska, and Lenape. These cultivars still showed high regeneration rates (87%–98% ) on media with 0.5 mg/1 IAA, and good regeneration rates were also achieved by the other three cultivars (48%, 94%, and 50% for Chieftain, Russet Burbank, and Shepody, respectively). Even with the single medium protocol (0.5% IAA without thiosulphate or thidiazuron), Désirée, Lenape, and Niska exhibited a regeneration rate of 98%. The use of petiole-with-leaflet explants could be ideal for the regeneration step of potato genetic transformation protocol because of their high regeneration efficiency and their small cut surface area forAgrobacterium elimination after co-incubation.     

Postprandial glycemia, insulinemia, and satiety responses in healthy subjects after whole grain rye bread made from different rye varieties. 1

Rye products typically induce low insulin responses and appear to facilitate glucose regulation. The objective of this study was to investigate differences in postprandial glucose, insulin, and satiety responses between breads made from five rye varieties. Breads made from whole grain rye (Amilo, Rekrut, Dankowski Zlote, Nikita, and Haute Loire Pop) or a white wheat bread (WWB) were tested in a randomized cross-over design in 14 healthy subjects (50 g available starch). Metabolic responses were also related to the composition of dietary fiber and bioactive compounds in the breads and to the rate of in vitro starch hydrolysis. The Amilo and Rekrut rye breads induced significantly lower insulin indices (II) than WWB. Low early postprandial glucose and insulin responses (tAUC 0-60 min) were related to higher amounts of caffeic, ferulic, sinapic, and vanillic acids in the rye breads, indicating that the phenolic acids in rye may influence glycemic regulation. All rye breads induced significantly higher subjective feelings of fullness compared to WWB. A low II was related to a higher feeling of fullness and a lower desire to eat in the late postprandial phase (180 min). The data indicate that some rye varieties may be more insulin-saving than others, possibly due to differences in dietary fiber, rate of starch hydrolysis, and bioactive components such as phenolic acids.     

<Emphasis Type="Italic">Streptococcus uberis</Emphasis>-specific T cells are present in mammary gland secretions of cows and can be activated to kill <Emphasis Type="Italic">S. uberis</Emphasis>

The presence, phenotype and function of Streptococcus uberis-specific T cells in the mammary gland secretion (MGS) and blood of cows exposed to S. uberis were assessed. MGS T cells in the udder were purified and incubated with autologous blood monocytes as antigen-presenting cells (APC). Most cows, irrespective of prior S. uberis infection status and lactation status, were shown to have S. uberis-specific T cells both in MGS and in the blood. When cells from a subgroup of cows were studied, it was found that the S. uberis-specific T cells produced high levels of interferon-gamma (IFN-γ), but low levels of interleukin-10 (IL-10). A high percentage of responding T cells were of the CD8 ⁺ memory (CD45RO) subset. T cells from the MGS specific for S. uberis were propagated from animals during the drying off period and expanded in vitro using interleukin-2 (IL-2) and S. uberis antigens. This led to the accumulation of T cells of the CD8 ⁺ subset bearing memory cell markers (CD45A ⁻ , CD45RO ⁺ ), which released high levels of IFN-γ. Four of the five T cell lines derived from the MGS of three animals had substantial direct killing activity towards S. uberis in vitro. It is concluded that there is an emergence of S. uberis-specific bactericidal T cells in the MGS of cows after infection or environmental exposure to S. uberis. Vaccines aimed at activating and expanding this T cell population in the mammary glands of cattle may offer an avenue for the prevention of mastitis caused by S. uberis.