Sediment organic matter in mountain lakes of north-western Slovenia and its stable isotopic signatures: records of natural and anthropogenic impacts

Sediment organic matter (OM) and its stable carbon and nitrogen isotopes were studied in 12 Slovenian mountain lakes in the Julian Alps. The lakes have different catchment areas and display a range of trophic states. Surface sediment atomic C/N ratios ranged from 8.4 to 13.2. Based on these C/N ratios, we concluded that autochthonous OM dominates in these lakes and constitutes approximately 65–92% of the total OM. Higher contributions of autochthonous OM sources were observed in lakes above the tree line. Relatively constant C/N ratios in the deeper sediments suggest that degradation processes are most intense in the upper few centimetres of the sediments and/or that remaining OM is relatively resistant to further degradation. Surface sediment δ¹³C and δ¹⁵N values ranged from −36.1 to −14.1‰ and from −5.2 to +1.1‰, respectively. In sediment cores from seven lakes, higher δ¹³C and lower δ¹⁵N values characterize oligotrophic lakes situated above the tree line, whereas the reverse is true for eutrophic lakes below the tree line that are also exposed to more anthropogenic impact. Carbon and nitrogen biogeochemical cycling differs considerably among the lakes. Stratigraphic shifts in carbon, total nitrogen, C/N ratios and stable C and N isotopes in cores record changes in inputs, and hence water column processes, as well as alterations in loading to the lakes. The stratigraphic variations are also the result of post-depositional diagenetic changes in the upper few centimetres of sediment. All the lakes show impacts from recent increases in atmospheric deposition of dissolved inorganic nitrogen. Application of sediment OM analysis thus proved to be useful to reconstruct paleoecological changes in sensitive mountain lake ecosystems that are either natural and/or anthropogenically derived.     

NMR Structure of the Human Prion Protein with the Pathological Q212P Mutation Reveals Unique Structural Features

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrP^C) conformer, denoted as infectious scrapie isoform or PrP^Sc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrP^Sc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β₂–α₂ loop region. This structure might provide new insights into the early events of conformational transition of PrP^C into PrP^Sc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β₂–α₂ loop and α₃ helix.     

Molecular phylogeny of reed beetles (Col., Chrysomelidae, Donaciinae): the signature of ecological specialization and geographical isolation

The Donaciinae consist of approximately 165 species predominantly occurring in the northern hemisphere. We analysed mitochondrial and nuclear DNA (COI, EF-1alpha) of 46 species to investigate their phylogeny and to discuss general topics in the context of insect herbivory (generalists versus specialists, ecological speciation). Phylogenetic reconstructions from various methodical approaches yielded very similar results. Clades corresponding to the traditional tribes/genera were recovered. Within the genus Donacia, species groups with characteristic host plant preference were identified. Estimated divergence times are discussed on the background of geological events. The origin of the Donaciinae is dated to 75-100 million years before present, after which they quickly diversified into the main groups. An initial split of those groups occurred in the Palaeocene. In the Eocene and Oligocene, major lineages specialized on certain host plants, where they radiated in the Miocene. This radiation was enforced by geographic isolation brought about by the final separation of America and Europe, after which there arose continental lineages within three larger species groups. In their evolution based on ecological specialization with a recently superimposed geographic isolation, the Donaciinae follow a pattern of specialists arising from generalists. Host plant shifts show that such a specialization is not necessarily an 'evolutionary dead-end'.     

Interaction with model membranes and pore formation by human stefin B: studying the native and prefibrillar states

Human stefin B, from the family of cystatins, is used as a model amyloidogenic protein in studies of the mechanism of amyloid fibril formation and related cytotoxicity. Interaction of the protein's prefibrillar oligomers/aggregates with predominantly acidic phospholipid membranes is known to correlate with cellular toxicity. In the present study, we measured membrane interaction of the prefibrillar and native states for three variants: the Y31 isoform studied previously, the wild-type protein and the G4R mutant; the latter is observed in progressive myoclonus epilepsy of type 1. In addition to using critical pressure and surface plasmon resonance, we assessed membrane permeabilization by calcein release and electrophysiological measurements. It was demonstrated for the first time that wild-type stefin B and the Y31 isoform are able to form pores in planar lipid bilayers, whereas G4R destroys the bilayer by a non pore-forming process. Similarities to other amyloidogenic proteins and the possible physiological implications of our findings are discussed.     

Nf2/merlin regulates hematopoietic stem cell behavior by altering microenvironmental architecture

Stem cell population size is highly regulated across species and tissue types, and alterations are associated with premature tissue failure or cancer. We assessed whether the tumor suppressor and mediator of cell contact inhibition Nf2/merlin plays a role in governing the hematopoietic stem cell pool by stem cell-autonomous or niche-determined processes. Hematopoietic stem cells in Nf2-deficient mice were increased in number and demonstrated a marked shift in location to the circulation. These changes were entirely dependent on changes in the microenvironment, with a marked increase in trabecular bone and marrow vascularity associated with increased VEGF, but without cell-autonomous alterations in stem cell characteristics. Nf2/merlin is critical for maintaining normal structure and function of the hematopoietic stem cell niche. It limits both bone and vascular components, and our model suggests that it thereby constrains stem cell number and position.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Local expression of enzymatically active class I β-1, 3-glucanase enhances symptoms of TMV infection in tobacco

Mutant tobacco plants deficient for class I beta-1,3-glucanase (GLU I) are decreased in their susceptibility to virus infection. This is correlated with delayed virus spread, a reduction in the size exclusion limit of plasmodesmata and increased cell-wall deposition of the beta-1,3-glucan callose. To further investigate a role of GLU I during cell-to-cell movement of virus infection, we inserted the GLU I coding sequence into TMV for overexpression in infected cells. Compared with the size of local lesions produced on plants infected with virus expressing either an enzymatically inactive GLU I or a frameshift mutant of the gene, the size of local lesions caused by infection with virus expressing active GLU I was consistently increased. Viruses expressing antisense GLU I constructs led to lesions of decreased size. Similar effects were obtained for virus spread using plants grown at 32 degrees C to block the hypersensitive response. Together, these results indicate that enzymatically active GLU I expressed in cells containing replicating virus can increase cell-to-cell movement of virus. This supports the view that GLU I induced locally during infection helps to promote cell-to-cell movement of virus by hydrolyzing callose. Moreover, our results provide the first direct evidence that a biological function of a plant beta-1,3-glucanase depends on its catalytic activity.     

Biological activity and characteristics of triamcinolone-acetonide formulated with the self-regulating drug carriers, Transfersomes®

Novel formulations of the halogenated corticosteroid, triamcinolone-acetonide, based on ultradeformable mixed lipid vesicles, Transfersomes®, are described. Their performance was tested in vivo using radioactive label measurements, to study the drug biodistribution, and murine ear edema, to determine the drug bioactivity. Sparse use of drug-loaded Transfersomes® on the skin ensures an almost exclusive delivery of triamcinolone-acetonide into the organ, thus arguably increasing the treatment safety. Delivery of triamcinolone-acetonide in the skin with ultradeformable vesicles prolongs the anti-inflammatory drug action several times compared to drug usage in a conventional crème or an ointment, the robustness of biological response for the former being at least identical to the latter. The required dose of Transfersome®-based triamcinolone-acetonide is also greatly reduced. The drug dose of 0.2 μg cm⁻² suppresses 75% of arachidonic acid-induced murine ear edema for at least 48 h. In contrast, a conventional formulation of triamcinolone-acetonide requires a 10-fold higher drug dosage to achieve a similar effect. In either case, increasing the applied corticosteroid amount delays the onset of anti-edema action.