Purification strategy for recombinant Phl p 6 is applicable to the natural allergen and yields biochemically and immunologically comparable preparations

The recombinant major grass pollen allergen Phl p 6 has been expressed with a N-terminal 6 x His-tag sequence and subsequently purified using nickel-chelating Sepharose. After cleavage of the tag-sequence, a second pass over the affinity chromatography revealed that even untagged rPhl p 6 bound tightly. In order to determine if that property is typical for Phl p 6, the natural allergen was purified in the same way starting with a grass pollen extract. Indeed, nPhl p 6 could be highly enriched in one step using nickel-chelating Sepharose. In addition to this new powerful purification method, the results provide further information in that the recombinant and natural allergens share a lot of properties, since biochemical characteristics are reflected in the purification strategies. The preparations of natural and recombinant Phl p 6 were used for comparative electrophoretic, chromatographic and immunological analysis which demonstrated high similarity.     

Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells

We have established a versatile method for studying the interaction of the oleosin gene product with oil bodies during oil body biogenesis in plants. Our approach has been to transiently express a green fluorescent protein (GFP)-tagged Arabidopsis oleosin gene fusion in tobacco leaf cells containing bona fide oil bodies and then to monitor oleosin-GFP expression using real-time confocal laser scanning microscopy. We show that normally non-oil-storing tobacco leaf cells are able to synthesize and then transport oleosin-GFP fusion protein to leaf oil bodies. Synthesis and transport of oleosin-GFP fusion protein to oil bodies occurred within the first 6 h posttransformation. Oleosin-GFP fusion protein exclusively associated with the endoplasmic reticulum and was trafficked in a Golgi-independent manner at speeds approaching 0.5 microm sec(-1) along highly dynamic endoplasmic reticulum positioned over essentially static polygonal cortical endoplasmic reticulum. Our data indicate that oil body biogenesis can occur outside of the embryo and that oleosin-GFP can be used to monitor early events in oil body biogenesis in real-time.     

Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

The IIIb isoform of fibroblast growth factor receptor 2 is required for proper growth and branching of pancreatic ductal epithelium but not for differentiation of exocrine or endocrine cells

Fibroblast growth factors (Fgfs) and their receptors have been implicated in embryonic pancreas development. Recently it was shown that Fgf10, a major ligand for the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2b), has an important regulatory role in early pancreas development. The aim of our study was to define the role of Fgfr2b in pancreas development by analyzing the phenotype of Fgfr2b (-/-) mice. Pancreases of Fgfr2b (-/-) embryos were noticeably smaller than the wild type littermates during embryogenesis, and pancreatic ductal branching as well as duct cell proliferation was significantly reduced. However, both exocrine and endocrine pancreatic differentiation occurred relatively normally. Exogenous addition of Fgfr2b ligands (Fgf7 and Fgf10) stimulated duct cell proliferation and inhibited endocrine cell differentiation in the ex vivo embryonic organ cultures of wild type pancreas. Our results thus suggest that Fgfr2b-mediated signaling plays a major role in pancreatic ductal proliferation and branching morphogenesis, but has little effect on endocrine and exocrine differentiation.     

Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.     

Seedling establishment in relation to microhabitat variation in a windthrow gap in a boreal Pinus sylvestris forest

Abstract. The characteristics of microhabitats of established Pinus sylvestris and Betula seedlings were studied in a small windthrow gap in a mature P. sylvestris-dominated forest in the Petkeljärvi National Park in eastern Finland. Seedlings were strongly clustered in disturbed microhabitats, particularly uprooting pits and mounds, formed by tree falls. They covered 3% of the 0.3.ha study area consisting of the gap and some of the forest edge. Although Betula occurred only as scattered individuals in the dominant canopy layer of the forest, it accounted for 30% of the seedlings found in the study area. Betula regeneration was almost completely restricted to pits and mounds, where 91% of the seedlings were found. Uprooting spots were also the most important regeneration microhabitats for Pinus, where 60% of the seedlings grew, even though the seedlings were found in other substrates as well, particularly on sufficiently decomposed coarse wood. Undisturbed field- and bottom-layer vegetation had effectively hindered tree seedling establishment, which emphasises the role of soil disturbance for regeneration. While the establishment of seedlings was found to be clearly determined by the availability of favourable regeneration microhabitats, the early growth of seedlings was affected by a complex interaction of environmental variables, including the type of microhabitat, radiation environment and interferences caused by competing seedlings and adjacent trees. In the most important regeneration microhabitats, i.e. in uprooting pits and on mounds, the distributions of the local elevations of Pinus and Betula seedlings were different. Pinus seedlings occurred closer to ground level, i.e. on the fringes of pits and lower on mounds, while Betula seedlings grew deeper in pits and higher on mounds. The position of the Betula seedlings indicate that they may have a competitive advantage over Pinus seedlings in the dense seedling groups occurring in uprooting spots. We suggest that this initial difference in Pinus and Betula establishment may affect the subsequent within-gap tree species succession and can, in part, explain the general occurrence of Betula in conifer-dominated boreal forests.     

Biomanipulation as an Application of Food-Chain Theory: Constraints, Synthesis, and Recommendations for Temperate Lakes

The aim of this review is to identify problems, find general patterns, and extract recommendations for successful biomanipulation. An important conclusion is that the pelagic food chain from fish to algae may not be the only process affected by a biomanipulation. Instead, this process should be viewed as the “trigger” for secondary processes, such as establishment of submerged macrophytes, reduced internal loading of nutrients, and reduced resuspension of particles from the sediment. However, fish reduction also leads to a high recruitment of young-of-the-year (YOY) fish, which feed extensively on zooplankton. This expansion of YOY the first years after fish reduction is probably a major reason for less successful biomanipulations. Recent, large-scale biomanipulations have made it possible to update earlier recommendations regarding when, where, and how biomanipulation should be performed. More applicable recommendations include (1) the reduction in the biomass of planktivorous fish should be 75% or more; (2) the fish reduction should be performed efficiently and rapidly (within 1–3 years); (3) efforts should be made to reduce the number of benthic feeding fish; (4) the recruitment of YOY fish should be reduced; (5) the conditions for establishment of submerged macrophytes should be improved; and (6) the external input of nutrients (phosphorus and nitrogen) should be reduced as much as possible before the biomanipulation. Recent biomanipulations have shown that, correctly performed, the method also achieves results in large, relatively deep and eutrophic lakes, at least in a 5-year perspective. Although repeated measures may be necessary, the general conclusion is that biomanipulation is not only possible, but also a relatively inexpensive and attractive method for management of eutrophic lakes, and in particular as a follow-up measure to reduced nutrient load. Received 14 April 1998; accepted 31 August 1998     

Regional and local drivers of macroinvertebrate assemblages in boreal springs

Aim To identify the most important environmental drivers of benthic macroinvertebrate assemblages in boreal springs at different spatial scales, and to assess how well benthic assemblages correspond to terrestrially derived ecoregions. Location Finland. Methods Benthic invertebrates were sampled from 153 springs across four boreal ecoregions of Finland, and these data were used to analyse patterns in assemblage variation in relation to environmental factors. Species data were classified using hierarchical divisive clustering (twinspan ) and ordinated using non‐metric multidimensional scaling. The prediction success of the species and environmental data into a priori (ecoregions) and a posteriori (twinspan ) groups was compared using discriminant function analysis. Indicator species analysis was used to identify indicator taxa for both a priori and a posteriori assemblage types. Results The main patterns in assemblage clusters were related to large‐scale geographical variation in temperature. A secondary gradient in species data reflected variation in local habitat structure, particularly abundance of minerogenic spring brooks. Water chemistry variables were only weakly related to assemblage variation. Several indicator species representing southern faunistic elements in boreal springs were identified. Discriminant function analysis showed poorer success in classifying sites into ecoregions based on environmental than on species data. Similarly, when classifying springs into the twinspan groups, classification based on species data vastly outperformed that based on environmental data. Main conclusions A latitudinal zonation pattern of spring assemblages driven by regional thermal conditions is documented, closely paralleling corresponding latitudinal patterns in both terrestrial and freshwater assemblages in Fennoscandia. The importance of local‐scale environmental variables increased with decreasing spatial extent. Ecoregions provide an initial stratification scheme for the bioassessment of benthic macroinvertebrates of North European springs. Our results imply that climate warming, landscape disturbance and degradation of spring habitat pose serious threats to spring biodiversity in northern Europe, especially to its already threatened southern faunistic elements.