Introduction of lanthanide(III) chelates to oligopeptides on solid phase

The synthesis of oligopeptide building blocks for the introduction of nonluminescent and luminescent lanthanide(III) chelates to the oligopeptide structure on the solid phase is described. The oligopeptide conjugates synthesized were used in DELFIA-based receptor binding assay (motilin) as well as in LANCE time-resolved fluorescence quenching assay (caspase-3).     

Nutrient concentration in shape Sphagna at increased N-deposition rates and raised atmospheric CO2 concentrations

Sphagnum fuscum, S. magellanicum, S. angustifolium and S. warnstorfii were treated with N deposition rates (0, 10, 30 and 100 kg ha^-1 a^-1) and with four atmospheric CO₂ concentrations (350, 700, 1000 and 2000 ppm) in greenhouse for 71–120 days. Thereafter, concentrations of total N, P, K, Ca and Mg in the capitulae of the Sphagna were determined. The response of each species to N deposition was related to ecological differences. With increasing N deposition treatments, moss N concentrations increased and higher N:P-ratios were found, the increase being especially clear at the highest N load. Sphagnum fuscum, which occupies ombrotrophic habitats, was the most affected by the increased nitrogen load and as a consequence the other elements were decreased. Oligotrophic S. magellanicum, wide nutrient status tolerant S. angustifolium and meso-eutrophic S. warnstorfii tolerated better increased N deposition, though there were increased concentrations of Ca and Mg in S. warnstorfii and Mg in S. magellanicum. Nitrogen and P concentrations decreased with raised CO₂ concentrations, except for S. magellanicum. This seems to be the first time this kind of response in nutrient concentrations to enhanced CO₂ concentration has been shown to exist in bryophytes. The concentration of K clearly decreased in S. fuscum as did the concentration of Mg in the other Sphagna with increasing CO₂. Sphagnum angustifolium and S. magellanicum, which are the less specialized species, were the least affected by the CO₂ treatments.     

Immobilization and bone structure in humans

Long-term immobilization is known to result in substantial bone loss. The present review examined the existing evidence for deterioration of bone structure during long-term disuse in humans. Paralysis due to spinal cord injury, long-term exposure to microgravity in space or tightly restricted mobility during bed rest provide reasonable models to assess the influence of immobilization on bone structure. Expectedly, the duration of immobilisation was the major determinant of bone loss, but irrespective of whether the skeletal unloading was due to irrecoverable paralysis, long-term spaceflight or bed rest, the mean pattern of structural deterioration of bone, mainly manifest as substantial cortical thinning and trabecular bone loss, was quite similar. However, skeletal responses to disuse can be highly variable between individuals. Apparently the relative decline in individual’s bone loading in relation to loading prior to immobilization accounts for inter-individual variation.     

Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of 'host range' mutants of E. coli K1 bacteriophages.     

SNP discovery by mismatch-targeting of Mu transposition

Single nucleotide polymorphisms (SNPs) represent a valuable resource for the mapping of human disease genes and induced mutations in model organisms. SNPs may become the markers of choice also for population ecology and evolutionary studies, but their isolation for non-model organisms with unsequenced genomes is often difficult. Here, we describe a rapid and cost-effective strategy to isolate SNPs that exploits the property of the bacteriophage Mu transposition machinery to target mismatched DNA sites and thereby to effectively detect polymorphic loci. To demonstrate the methodology, we isolated 164 SNPs from the unsequenced genome of the Glanville fritillary butterfly (Melitaea cinxia), a much-studied species in population biology, and we validated 24 of them. The strategy involves standard molecular biology techniques as well as undemanding MuA transposase-catalyzed in vitro transposition reactions, and it is applicable to any organism.     

Impacts of nutrient enrichment and sediment on phytoplankton community structure in the northern Baltic Sea

A three-week mesocosm experiment was conducted in order to study the effects of bottom sediment and nutrient enrichment on phytoplankton and zooplankton community structure in the Archipelago Sea, northern Baltic Sea. The transparent polyethylene enclosures included the whole water column and varied in volume from 30 to 40 m³. There were two types of enclosures: some with natural sediment as a bottom and others with a plastic bottom. The experiment was a 2 × 2 factorial design with presence of sediment and nutrient enrichment as treatment factors. Both the sediment presence and nutrient enrichment significantly increased water nutrient concentrations and the rate of primary production. However, external nutrient enrichment and the presence of sediment stimulated the growth of different phytoplankton groups, indicating that the effect of sediment was not related to nutrient fluxes alone, but involved more complex interactions. External nutrient enrichment was primarily channelled to picoplanktonic cyanobacteria, the biomass of which increased four- to fivefold due to enrichment. The presence of sediment increased the biomass of cryptophytes, chrysophytes and prasinophytes, but decreased the biomass of N₂-fixing cyanobacteria. Zooplankton biomass increased during the experiment, but was not affected by the treatments. The study shows that sediment plays a significant role in phytoplankton dynamics, underlining the importance of including sediment in shallow-water mesocosm experiments. Handling editor: J. Padisak     

Biomass,composition and activity of organism assemblages along a salinity gradient in sea ice subjected to river discharge in the Baltic Sea

A study was undertaken to examine the activity and composition of the seasonal Baltic Sea land-fast sea-ice biota along a salinity gradient in March 2003 in a coastal location in the SW coast of Finland. Using a multi-variable data set, the less well-known algal and protozoan communities, and algal and bacterial production in relation to the physical and chemical environment were investigated. Also, the first coincident measurements of bacterial production and dissolved organic matter (DOM) in a sea-ice system are reported. Communities in sea ice were clearly autotrophy-dominated with algal biomass representing 79% of the total biomass. Protozoa and rotifers made up 18% of biomass in the ice and bacteria only 3%. Highest biomasses were found in mid-transect bottom ice. Water column assemblages were clearly more heterotrophic: 39% algae, 12% bacteria and 49% for rotifers and protozoa. Few significant correlations existed between DOM and bacterial variables, reflecting the complex origin of ice DOM. Dynamics of dissolved organic carbon, nitrogen and phosphorus (DOC, DON and DOP) were also uncoupled. A functional microbial loop is likely to be present in the studied ice. Existence of an under-ice freshwater plume affects the ecosystem functioning: Under-ice water communities are influenced directly by river-water mixing, whereas the ice system seems to be more independent—the interaction mainly taking place through the formation of active bottom communities.     

Solid-supported 2'-O-glycoconjugation of oligonucleotides by azidation and click reactions

2'-O-[(2-Bromoethoxy)methyl]cytidine and 2'-O-[(2-azidoethoxy)methyl]cytidine have been prepared and introduced as appropriately protected 3'-phosphoramidite (1) and 3'-(H-phosphonate) (2) building blocks, respectively, into 2'-O-methyl oligoribonucleotides. The support-bound oligonucleotides were subjected to two consecutive conjugations with alkynyl-functionalized monosaccharides. The first saccharide was introduced by a Cu(I) promoted click reaction with 2 and the second by azidation of the 2-bromoethoxy group of 1 followed by the click reaction. The influence of the 2'-glycoconjugations on hybridization with DNA and 2'-O-methyl RNA targets was studied. Two saccharide units within a 15-mer oligonucleotide had a barely noticeable effect on the duplex stability, while introduction of a third one moderately decreased the melting temperature.     

Attenuated expression of tenascin-c in ovalbumin-challenged STAT4-/- mice

Background

Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity.

Methods

Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro.

Results

OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings.

Conclusions

Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.