Effects of T3 and rearing temperature on growth and skeletal myosin heavy chain isoform transition during early development in the salmonid Salvelinus alpinus (L.)

Rearing of 1-year-old Arctic charr (Salvelinus alpinus) at 12°C, as well as the administration of 50 or 75 mgT₃/kg feed, accelerated the neonatal to adult fast myosin heavy chain transition, but the effect of temperature was more dramatic than the effect of T₃ administration. The endogenous plasma levels of T₃ in charrs reared at 12°C were higher than those of analogous groups reared at natural temperature, which in the period under study was between 0.5 and 12°C. As in other species, T₃ seemed to play a role in the regulation of the neonatal to adult fast myosin isoform transition by down-regulating the levels of the neonatal and increasing the levels of an adult fast myosin heavy chain. Temperature seemed to accelerate this transition at least, but not only, by inducing an increase in the endogenous levels of T₃ in the Arctic charr.     

Glycan modification of a thermostable recombinant (1-3,1-4)-β-glucanase secreted fromSaccharomyces cerevisiae is determined by strain and culture conditions

High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)--glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1_p) and theBacillus macerans (1-3,1-4)--glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man_8-13GlcNAc₂, although only traces of Man₉GlcNAc₂ were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y. Abbreviations: AMY,B. amyloliquefaciens (1-3,1-4)--glucanse; MAC,B. macerans (1-3,1-4)--glucanase H(A16-M), H(A36-M), H(A78-M),H(A107-M) and H(A152-M), hybrid (1-3,1-4)--glucanases containing 16, 36, 78, 107 and 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC; similar enzyme abbreviations followed by Y, e.g. H(A16-M)-Y, denote the enzymes secreted from yeast cells; PCR, polymerase chain reaction; PGK_p, yeast 3-phosphoglycerate kinase promoter; PRB1_p, yeast protease B promoter; LB, Luria-Bertani medium; SC, minimal medium; CNBr, cyanogen bromide; Endo H_f, endoglycosidase H fusion protein; PNGase F, peptide:N-glycosidase F; HPAEC; high pH anion exchange chromatography; HVE, high voltage paper electrophoresis; CPY, yeast carboxypeptidase Y.     

High-resolution mapping by YAC fragmentation of a 2.5-Mb Xp22 region containing the human RS, KFSD and CLS disease genes

The disease loci for X-linked Retinoschisis (RS), Keratosis follicularis spinulosa decalvans (KFSD), and Coffin-Lowry syndrome (CLS) have been localized to the same, small region in Xp22 on the human X Chromosome (Chr). To generate a high-resolution map of the available contig in this area, we have used the YAC fragmentation vectors pBP108/ADE2 and pBP109/ADE2 and generated fragmented YACs from a 2.5-Mb YAC (y939H7) spanning the mentioned disease gene candidate regions. Forty-seven fragmented YACs were generated and analyzed, ranging in size from 170 kb to over 2400 kb. The resulting YAC fragmentation panel was used to construct a detailed restriction map of the region and has been used to bin clones and markers. As a deletion panel, it will present a valuable resource for further mapping. Received: 31 December 1996 / Accepted: 22 February 1997     

C andH NMR spectroscopy as a tool in the configurational analysis of iridoid glucosides

The ¹³C NMR data of 51 iridoid glucosides or glucoside acetates are tabulated. The collection includes 20 pairs of C-6, C-7 or C-8 epimers. Three parameters in using the data for the configurational assignment of 6-O-substituents are given. The chemical shift for C-9 in a range of substituted compounds is shown to be numerically related to the stereochemistry at C-8. This allows the determination of the configuration at this centre for most types of substitution patterns by calculation of the C-9 shift using increments for each substituent. Such increments are given for 25 substituents in three different solvents. A method for simulation of spectra of unknown iridoid glucosides is presented. By this method, the structures of five novel iridoid glucosides have been elucidated, and that of tecomoside has been revised. The methods used to assign the configurations to C-6 and C-8 epimeric iridoid glucosides by ¹H NMR spectroscopy are discussed and a table with selected data is presented. It is suggested that the structures in the literature for ajugol and myoporoside should be interchanged. Consequently, Horeau's method has failed in these instances. Finally, the differences in the ¹³C NMR spectra of pairs of C-6 and C-8 epimeric iridoid glucosides have been interpreted as originating from cis/trans-interactions.     

Ceruloplasmin Polymorphism and Parentage Test in Hereford Cattle

In recent years parentage control by means of blood grouping tests (blood and protein systems) has been required for bulls to be registered in the Danish Hereford Herd Book. Because the Hereford breed shows less variation in the blood and protein systems, the probability of excluding an incorrectly stated bull (or cow) is estimated to be some 15 % lower in Hereford than in Danish dairy breeds (RDM, SDM and Jersey).     

Pitfalls in the sample preparation and analysis of N-acylethanolamines

N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.