Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species.

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

Molecular characterisation of a membrane-bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis.

Galactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP-mannose dependent (1-->4)-beta-D-mannan synthase and a UDP-galactose dependent (1-->6)-alpha-D-galactosyltransferase. The statistical distribution of galactosyl substituents along the mannan backbone, and the degree of galactose substitution of the primary product of galactomannan biosynthesis appear to be regulated by the specificity of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identification on gels of a putative 51 kDa galactosyltransferase protein, and the isolation, cloning and sequencing of the corresponding cDNA. The solubilised galactosyltransferase has an absolute requirement for added acceptor substrates. Beta-(1-->4)-linked D-manno-oligosaccharides with chain lengths greater than or equal to 5 acted as acceptors, as did galactomannans of low to medium galactose-substitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also confirmed the identity of the galactosyltransferase by inserting the cDNA in frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an AOX promoter and the yeast alpha secretion factor and observing the secretion of galactomannan alpha-galactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane-spanning helix, was expressed.

Parathyroid hormone inhibits collagen synthesis at both ribonucleic acid and protein levels in rat osteogenic sarcoma cells.

Cells of the clonal rat osteogenic sarcoma cell line, UMR 106-01, were used to investigate the regulation of collagen synthesis by PTH in osteoblastic cells. Monolayer cultures of cells were labeled with [3H] proline in order to determine both collagen type and rates of production. Analysis of labeled extracellular polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that UMR 106-01 cells synthesized predominantly type I collagen, accounting for 45.48 +/- 2.09% of the radioactivity incorporated into total protein. After 24-h treatment with bovine PTH (1-34, 10(-8) M), collagen synthesis (i.e. collagenase-digestible protein) was decreased to 29.45 +/- 1.39% of total protein production. This decrease was first observed 12 h after addition of hormone and greatest inhibition was achieved at 24 h. The effect of PTH was dose dependent, with half-maximal inhibition of collagen synthesis occurring at 5 x 10(-10) M after 24-h treatment. In contrast, when steady state levels of mRNA for type I collagen chains were examined by Northern blot analysis, the concentration of PTH that reduced collagen synthesis by 35-45% (10(-8) M), caused a net decrease of approximately 80-96% in the number of procollagen transcripts; a small reduction in beta-actin mRNA levels was also observed. The effect of the hormone on procollagen message level was dose dependent, with significant inhibition observed at 10(-10) M PTH and, as with collagen synthesis, maximal after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Imperforate hymen with hematocolpos.

Presented are two cases of imperforate hymen with hematocolpos seen in a pediatric emergency department (ED) during a seven-month period. The first case presented with abdominal pain, urinary obstruction, and constipation on initial visit. The diagnosis was not made on the initial presentation. The patient was seen a second time, and final diagnosis was not made until a third visit to the pediatric outpatient clinic. The second case presented with syncope and bilateral lower abdominal pain. Ultrasound and subsequent surgery confirmed the physical findings in the ED of imperforate hymen and hematocolpos. Both patients underwent hymenectomy, and they have experienced no further symptoms.

The Late-Weichselian Flora of the Isle of Man.

The last glacial deposits of the Ballaugh-Kirkmichael area in the north-east of the Isle of Man have been investigated by analysis of pollen and macroscopic fossils and by radiocarbon dating. Assemblages totalling over 160 taxa of vascular plants and mosses have been recorded from strata referred to Late-Weichselian zones I, II and III. Among the most noteworthy species are 46 not now living on the island; these include Dianthus deltoides, Juncus balticus, Lychnis viscaria, Ranunculus hyperboreus, Sibbaldia procumbens, Meesia tristicha, Helodium blandowii and Polytrichum norvegicum. The vegetation comprised a great diversity of communities of open, largely calcareous grassland, snow beds, mires both base-rich and base-poor, flushes, freshwater, inundated flats and calcareous dunes. Saline conditions are indicated by Glaux maritima and Triglochin maritima. Trees were represented only by Betula and the taller shrubs by Juniperus and Salix.