Morphological variation between populations of the expanding ectoparasitic deer ked Lipoptena cervi (Diptera: Hippoboscidae) in Fennoscandia

We examined morphological and genetic differences among Fennoscandian deer ked (Lipoptena cervi L, Hippoboscidae) populations with varying expansion history: the eastern population (Finland) has expanded rapidly, whereas the western population is divided into an old and relatively stationary sub‐population in Sweden and a newly established and more expansive sub‐population in Norway. The genetic analysis suggests that the distinct populations represent a single species. Individuals from expansive populations were characterized by a large body size, relatively large and robust thorax shape, and wing shape with an exaggerated basal posterior margin. Yet, there was no among population variation in relative wing size or its elongated shape after variation in overall size was controlled for. Although certain size and shape variables showed thermal sensitivity, the degree of plasticity did not differ between the populations. In general, we observed that shape is more sensitive to external thermal conditions at the pupal stage than size per se, with the thermal sensitivity of the latter depending on the trait under examination. We conclude that the possible adaptive value of morphological differences relies on variation in survival during the off‐host life stages or short‐distance flight to reach a susceptible host instead of long‐distance dispersal ability.     

Intracellularly truncated human alpha2B-adrenoceptors: stable and functional GPCRs for structural studies

All three alpha2-adrenoceptor subtypes have a long third intracellular loop (3i), which is conserved by overall size and charge-hydrophobic properties but not by amino acid sequence similarity. These properties must be relevant for function and structure, because they have been preserved during hundreds of millions of years of evolution. The contribution of different loop portions to agonist/antagonist binding properties and G protein coupling of the human alpha2B-adrenoceptor (alpha2B-AR) was investigated with a series of 3i truncated constructs (delta3i). We used a variety of agonists/antagonists in competition binding assays. We stimulated alpha2B-AR delta3i with various agonists and measured [35S]GTPgammaS binding in isolated cell membranes with or without antagonist inhibition. We also evaluated the ability of oligopeptides, analogous to the amino and carboxyl terminal parts of 3i, to promote G protein activation, monitored with the [35S]GTPgammaS assay. Our results reveal that the carboxyl end residues of 3i, R360(6.24) to V372(6.36), are important for Gi/Go protein activation. Deletions in regions from G206(5.72) to R245(5.110) altered the binding of some alpha2B-AR agonists, indicating that agonist binding is dependent on the conformation of the 3i domain, possibly through the involvement of G protein interactions. The truncated receptor constructs may be more stable on purification and thus be useful for structural characterization of alpha2B-AR.     

Production and purification of recombinant human α2C2 adrenergic receptor using Saccharomyces cerevisiae

The objective is to generate milligram quantities of recombinant human ₂C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.     

Ascorbic acid metabolism during bilberry (Vaccinium myrtillus L.) fruit development

Bilberry (Vaccinium myrtillus L.) possesses a high antioxidant capacity in berries due to the presence of anthocyanins and ascorbic acid (AsA). Accumulation of AsA and the expression of the genes encoding the enzymes of the main AsA biosynthetic route and of the ascorbate-glutathione cycle, as well as the activities of the enzymes involved in AsA oxidation and recycling were investigated for the first time during the development and ripening of bilberry fruit. The results showed that the AsA level remained relatively stable during fruit maturation. The expression of the genes encoding the key enzymes in the AsA main biosynthetic route showed consistent trends with each other as well as with AsA levels, especially during the first stages of fruit ripening. The expression of genes and activities of the enzyme involved in the AsA oxidation and recycling route showed more prominent developmental stage-dependent changes during the ripening process. Different patterns of activity were found among the studied enzymes and the results were, for some enzymes, in accordance with AsA levels. In fully ripe berries, both AsA content and gene expression were significantly higher in skin than in pulp.     

Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots

Buds and shoot tips of wild bilberry (Vaccinium myrtillus L.) and lingonberry (V. vitis-idaea L.) plants were cultured on a modified MS medium containing N⁶-isopentenyladenine (2iP), 9.8–78.4 μM, in order to study the effect of the 2iP-concentration on the initiation of growth. The experiment was first performed in the autumn and repeated in the spring to determine the influence of season on growth initiation. To optimise rooting, three different rooting treatments were tested for the bilberry and lingonberry microshoots. Shoots were rooted either in vitro with 0.49 μM IBA (indole-3-butyric acid) or ex vitro, incubating microshoots in 2.07 mM KIBA-solution (potassium salt of IBA) before planting, or microshoots were planted directly on peat without exogenous auxin. The best 2iP concentration for the initiation of the growth for bilberry was 49.2 μM and for lingonberry 24.6 μM. It was observed that increasing the 2iP concentration at the growth initiation stage increased the number of brownish explants both in bilberry and in lingonberry microcultures. Spring was a considerably better time than autumn for the initiation of new growth, for both species. The results of the rooting test showed that the KIBA-treatment before planting on peat increases rooting efficiency in both bilberry and lingonberry. This revised version was published online in June 2006 with corrections to the Cover Date.     

cDNA blotting offers an alternative method for gene expression studies

Northern analysis of bilberry fruit RNA using a non-radioactive detection system proved to be extremely difficult. To overcome this problem, we used cDNA instead of RNA for the blotting step. The RNA was translated to cDNA directly after isolation. The cDNA was then separated by electrophoresis, stained with ethidium bromide, and blotted onto a nylon membrane by Southern transfer. Non-radioactive detection by digoxigenin-dUTP was then possible. This method resolves several problems associated with northern blotting and is especially efficient when applied to recalcitrant plant material.     

DNA isolation methods for medicinal and aromatic plants

Several protocols described for plant DNA isolation fail to produce good quality DNA from medicinal herbs and aromatic plants. These plants contain exceptionally high amounts of secondary metabolites that interfere with DNA isolation. To address this problem, we developed 2 DNA isolation methods for sundew and tarragon that produce DNA suitable for molecular biological applications. One of the methods also is applicable for milfoil and Siberian ginseng.