A Novel Bacterial Enzyme with d-Glucuronyl C5-epimerase Activity

Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of d-glucuronic acid to its C5-epimer l-iduronic acid, which is essential for the function of heparan sulfate. Although l-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic d-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with d-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human d-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial d-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.     

Establishment and Early Growth of Willow at Different Levels of Weed Competition and Nitrogen Fertilization

To evaluate the effects of weed competition and nitrogen fertilization on the early growth performance of willow, cuttings of the clone Tora (Salix schwerinii x S. viminalis) were planted in buckets together with model weeds (spring barley or white mustard) sown 15, 26, and 30 days after willow planting. The buckets were fertilized with 30 or 90 kg N ha^?1. Willow with weeds sown after 15 days produced less biomass and smaller leaf area and had a lower maximum shoot height compared to willow planted without weeds and willow with weeds sown after 26 or 30 days. Fertilization with 90 kg N ha^?1 gave higher willow biomass production in willow with weeds sown after 26 or 30 days. Type of model weed had no effect on willow performance. Weed biomass and maximum shoot height were higher in weeds planted without willows compared to the willow-weed mixtures. A high nitrogen level gave more weed biomass when planted without willows and in the willow-weed mixture with weeds sown after 15 days. We conclude that for the given high density of willow, competition from weeds emerging soon after willow planting had strong effect on early production. Furthermore, if there is a risk of weed infestation, fertilization should be delayed.     

Comparative polytene chromosome maps of <Emphasis Type="Italic">D. montana</Emphasis> and <Emphasis Type="Italic">D. virilis</Emphasis>

Chromosomal inversion polymorphism was characterized in Finnish Drosophila montana populations. A total of 14 polymorphic inversions were observed in Finnish D. montana of which nine had not been described before. The number of polymorphic inversions in each chromosome was not significantly different from that expected, assuming equal chance of occurrence in the euchromatic genome. There was, however, no correlation between the number of polymorphic inversions and that of fixed inversions in each chromosome. Therefore, a simple neutral model does not explain the evolutionary dynamics of inversions. Furthermore, in contrast to results obtained by others, no significant correlation was found between the two transposable elements (TEs) Penelope and Ulysses and inversion breakpoints in D. montana. This result suggests that these TEs were not involved in the creation of the polymorphic inversions seen in D. montana. A comparative analysis of D. montana and Drosophila virilis polytene chromosomes 4 and 5 was performed with D. virilis bacteriophage P1 clones, thus completing the comparative studies of the two species.     

Pyrone and pyridone compounds in the liquid culture of Physisporinus sanguinolentus

Chromatographic separation of the liquid culture filtrate of the basidiomycete fungus Physisporinus sanguinolentus has yielded three new compounds viz., 2-methyl-4-pyrone, 2-methyl-5,6-dihydro-4-pyrone and the pyridone form of 4-hydroxy-2-methylpyridine, together with the known triacetic acid lactone, the sesquiterpene dialdehyde merulidial and a derivative of merulidial. Their structures were elucidated by spectroscopic analysis and by comparison to literature data and a synthetic sample. One of the compounds, merulidial, was shown to inhibit the germination of spores and the hyphal growth of the wood-rotting basidiomycete Heterobasidion annosum and the saprophytic mould Cladosporium cucumerinum.     

Short-term intermittent exposure to diazoxide improves functional performance of beta-cells in a high-glucose environment

Prolonged periods of "beta-cell rest" exert beneficial effects on insulin secretion from pancreatic islets subjected to a high-glucose environment. Here, we tested for effects of short-term intermittent rest achieved by diazoxide. Rat islets were cultured for 48 h with 27 mmol/l glucose alone, with diazoxide present for 2 h every 12 h or with continuous 48-h presence of diazoxide. Both protocols with diazoxide enhanced the postculture insulin response to 27 mmol/l glucose, to 200 mumol/l tolbutamide, and to 20 mmol/l KCl. Intermittent diazoxide did not affect islet insulin content and enhanced only K(ATP)-dependent secretion, whereas continuous diazoxide increased islet insulin contents and enhanced both K(ATP)-dependent and -independent secretory effects of glucose. Intermittent and continuous diazoxide alike increased postculture ATP-to-ADP ratios, failed to affect [(14)C]glucose oxidation, but decreased oxidation of [(14)C]oleate. Neither of the two protocols affected gene expression of the ion channel-associated proteins Kir6.2, sulfonylurea receptor 1, voltage-dependent calcium channel-alpha1, or Kv2.1. Continuous, but not intermittent, diazoxide decreased significantly mRNA for uncoupling protein-2. A 2-h exposure to 20 mmol/l KCl or 10 mumol/l cycloheximide abrogated the postculture effects of intermittent, but not of continuous, diazoxide. Intermittent diazoxide decreased islet levels of the SNARE protein SNAP-25, and KCl antagonized this effect. Thus short-term intermittent diazoxide treatment has beneficial functional effects that encompass some but not all characteristics of continuous diazoxide treatment. The results support the soundness of intermittent beta-cell rest as a treatment strategy in type 2 diabetes.     

Quantitative analysis of micro-CT imaging and histopathological signatures of experimental arthritis in rats

Micro-computed tomographic (micro-CT) imaging provides a unique opportunity to capture 3-D architectural information in bone samples. In this study of pathological joint changes in a rat model of adjuvant-induced arthritis (AA), quantitative analysis of bone volume and roughness were performed by micro-CT imaging and compared with histopathology methods and paw swelling measurement. Micro-CT imaging of excised rat hind paws (n = 10) stored in formalin consisted of approximately 600 30-mum slices acquired on a 512 x 512 image matrix with isotropic resolution. Following imaging, the joints were scored from H&E stained sections for cartilage/bone erosion, pannus development, inflammation, and synovial hyperplasia. From micro-CT images, quantitative analysis of absolute bone volumes and bone roughness was performed. Bone erosion in the rat AA model is substantial, leading to a significant decline in tarsal volume (27%). The result of the custom bone roughness measurement indicated a 55% increase in surface roughness. Histological and paw volume analyses also demonstrated severe arthritic disease as compared to controls. Statistical analyses indicate correlations among bone volume, roughness, histology, and paw volume. These data demonstrate that the destructive progression of disease in a rat AA model can be quantified using 3-D micro-CT image analysis, which allows assessment of arthritic disease status and efficacy of experimental therapeutic agents.     

Identification of the antigenic epitopes in staphylococcal enterotoxins A and E and design of a superantigen for human cancer therapy

Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.