Interaction between Oligomers of Stefin B and Amyloid-�� in Vitro and in Cells

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-β-(1–40) peptide (Aβ). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Aβ is oligomer specific. The dimers and tetramers of stefin B, which bind Aβ, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Aβ fibril formation. When expressed in cultured cells, stefin B co-localizes with Aβ intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Aβ epitope. Thus, stefin B is another APP/Aβ-binding protein in vitro and likely in cells.     

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid‐β peptide 1‐42 but not amylin and insulin fibrils can grow under quiescent conditions

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides – Alzheimer's amyloid‐β (Aβ) 1‐40 and 1‐42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aβ₄₀ substantially decreased, whereas the fibrillization of Aβ₄₂ peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Long-Range Gene Flow and the Effects of Climatic and Ecological Factors on Genetic Structuring in a Large,Solitary Carnivore: The Eurasian Lynx

Due to their high mobility, large terrestrial predators are potentially capable of maintaining high connectivity, and therefore low genetic differentiation among populations. However, previous molecular studies have provided contradictory findings in relation to this. To elucidate patterns of genetic structure in large carnivores, we studied the genetic variability of the Eurasian lynx, Lynx lynx throughout north-eastern Europe using microsatellite, mitochondrial DNA control region and Y chromosome-linked markers. Using SAMOVA we found analogous patterns of genetic structure based on both mtDNA and microsatellites, which coincided with a relatively little evidence for male-biased dispersal. No polymorphism for the cytochrome b and ATP6 mtDNA genes and Y chromosome-linked markers were found. Lynx inhabiting a large area encompassing Finland, the Baltic countries and western Russia formed a single genetic unit, while some marginal populations were clearly divergent from others. The existence of a migration corridor was suggested to correspond with distribution of continuous forest cover. The lowest variability (in both markers) was found in lynx from Norway and Białowieża Primeval Forest (BPF), which coincided with a recent demographic bottleneck (Norway) or high habitat fragmentation (BPF). The Carpathian population, being monomorphic for the control region, showed relatively high microsatellite diversity, suggesting the effect of a past bottleneck (e.g. during Last Glacial Maximum) on its present genetic composition. Genetic structuring for the mtDNA control region was best explained by latitude and snow cover depth. Microsatellite structuring correlated with the lynx''s main prey, especially the proportion of red deer (Cervus elaphus) in its diet. Eurasian lynx are capable of maintaining panmictic populations across eastern Europe unless they are severely limited by habitat continuity or a reduction in numbers. Different correlations of mtDNA and microsatellite population divergence patterns with climatic and ecological factors may suggest separate selective pressures acting on males and females in this solitary carnivore.     

Direct Compression of Cellulose and Lignin Isolated by a New Catalytic Treatment

Tablet compression of softwood cellulose and lignin prepared by a new catalytic oxidation and acid precipitation method were investigated and compared with the established pharmaceutical direct compression excipients. Catalytic pretreated softwood cellulose (CPSC) and lignin (CPSL) were isolated from pine wood (Pinus sylvestris). The compaction studies were carried out with an instrumented eccentric tablet machine. The plasticity and elasticity of the materials under compression were evaluated using force-displacement treatment and by determining characteristic plasticity (PF) and elasticity (EF) factors. With all biomaterials studied, the PF under compression decreased exponentially as the compression force increased. The compression force applied in tablet compression did not significantly affect the elasticity of CPSC and microcrystalline cellulose (MCC) while the EF values for softwood lignins increased as compression force increased. CPSL was clearly a less plastically deforming and less compactable material than the two celluloses (CPSC and MCC) and hardwood lignin. CPSL presented deformation and compaction behaviour almost identical to that of lactose monohydrate. In conclusion, the direct tablet compression behaviour of native lignins and celluloses can greatly differ from each other depending on the source and isolation method used.     

Upper Ordovician sequences of western Estonia

The Upper Ordovician (uppermost Caradoc-Ashgill) section of western Estonia consists of a series of seven open-shelf carbonate sequences. Depositional facies grade laterally through a series of shelf-to-basin facies belts: grain-supported facies (shallow shelf), mixed facies (middle shelf), mud-supported facies (deep shelf and slope) and black shale facies (basin). Locally, a stromatactis mud mound occurs in a middle-to-deep shelf position. Shallow-to-deep shelf facies occur widely across the Estonian Shelf and grade laterally through a transitional (slope) belt into the basinal deposits of the Livonian Basin.

Each sequence consists of a shallowing-upward, prograding facies succession. Sequences 1 (Upper Nabala Stage) and 2 (Vormsi Stage) record step-wise drowning of underlying shelf units (lower Nabala) that culminated in the deposition of the most basinal facies (Fjäcka Shale) in the Livonian Basin. Sequences 3–6 comprise the overlying Pirgu Stage and record the gradual expansion of shallow and middle-shelf facies across the Estonian Shelf. The Porkuni Stage (sequence 7) is bracketed by erosional surfaces and contains the shallowest-water facies of the preserved strata. The uppermost part of the section (Normalograptus persculptus biozone) is restricted to the Livonian Basin, and includes redeposited carbonate and siliciclastic grains; it is the lowstand systems tract of the lowest Silurian sequence 8. Sequence 7 and the overlying basinal redeposited material (i.e., the lowstand of sequence 8) correspond to the latest Ordovician (Hirnantian) glacial interval, and the bracketing unconformities are interpreted as the widely recognized early and late Hirnantian glacial maximums.

The sequences appear correlative to Upper Ordovician sequences in Laurentia. Graptolite biozones indicated that the Estonian sequences are equivalent to carbonate ramp sequences in the western United States (Great Basin) and mixed carbonate-siliciclastic sequences in the eastern United States (Appalachian Basin–Cincinnati Arch region). These correlations indicate a strong eustatic control over sequence development despite the contrasting tectonic settings of these basins.     

Mitogenetic structure of brown bears (Ursus arctos L.) in northeastern Europe and a new time frame for the formation of European brown bear lineages

We estimated the phylogenetic relationships of brown bear maternal haplotypes from countries of northeastern Europe (Estonia, Finland and European Russia), using sequences of mitochondrial DNA (mtDNA) control region of 231 bears. Twenty-five mtDNA haplotypes were identified. The brown bear population in northeastern Europe can be divided into three haplogroups: one with bears from all three countries, one with bears from Finland and Russia, and the third composed almost exclusively of bears from European Russia. Four haplotypes from Finland and European Russia matched exactly with haplotypes from Slovakia, suggesting the significance of the current territory of Slovakia in ancient demographic processes of brown bears. Based on the results of this study and those from the recent literature, we hypothesize that the West Carpathian Mountains have served either as one of the northernmost refuge areas or as an important movement corridor for brown bears of the Eastern lineage towards northern Europe during or after the last ice age. Bayesian analyses were performed to investigate the temporal framework of brown bear lineages in Europe. The molecular clock was calibrated using Beringian brown bear sequences derived from radiocarbon-dated ancient samples, and the estimated mutation rate was 29.8% (13.3%-47.6%) per million years. The whole European population and Western and Eastern lineages formed about 175,000, 70,000 and 25,000 years before present, respectively. Our approach to estimating the time frame of brown bear evolution demonstrates the importance of using an appropriate mutation rate, and this has implications for other studies of Pleistocene populations.     

A European Concern? Genetic Structure and Expansion of Golden Jackals (Canis aureus) in Europe and the Caucasus

In the first continent-wide study of the golden jackal (Canis aureus), we characterised its population genetic structure and attempted to identify the origin of European populations. This provided a unique insight into genetic characteristics of a native carnivore population with rapid large-scale expansion. We analysed 15 microsatellite markers and a 406 base-pair fragment of the mitochondrial control region. Bayesian-based and principal components methods were applied to evaluate whether the geographical grouping of samples corresponded with genetic groups. Our analysis revealed low levels of genetic diversity, reflecting the unique history of the golden jackal among Europe’s native carnivores. The results suggest ongoing gene flow between south-eastern Europe and the Caucasus, with both contributing to the Baltic population, which appeared only recently. The population from the Peloponnese Peninsula in southern Greece forms a common genetic cluster with samples from south-eastern Europe (ΔK approach in STRUCTURE, Principal Components Analysis [PCA]), although the results based on BAPS and the estimated likelihood in STRUCTURE indicate that Peloponnesian jackals may represent a distinct population. Moreover, analyses of population structure also suggest either genetic distinctiveness of the island population from Samos near the coast of Asia Minor (BAPS, most STRUCTURE, PCA), or possibly its connection with the Caucasus population (one analysis in STRUCTURE). We speculate from our results that ancient Mediterranean jackal populations have persisted to the present day, and have merged with jackals colonising from Asia. These data also suggest that new populations of the golden jackal may be founded by long-distance dispersal, and thus should not be treated as an invasive alien species, i.e. an organism that is “non-native to an ecosystem, and which may cause economic or environmental harm or adversely affect human health”. These insights into the genetic structure and ancestry of Baltic jackals have important implications for management and conservation of jackals in Europe. The golden jackal is listed as an Annex V species in the EU Habitats Directive and as such, considering also the results presented here, should be legally protected in all EU member states.     

Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

Aggregation of Aβ peptides into amyloid plaques is considered to trigger the Alzheimer’s disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aβ aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H₂O₂. The fibrillization of Aβ with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aβ with oxidized Met35 in the presence of H₂O₂, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ–Cu(II) complex.     

Organization and assembly of metal-thiolate clusters in epithelium-specific metallothionein-4

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd(2+) and Co(2+) ions. Both the (113)Cd NMR studies of (113)Cd(7)MT-4 and the spectroscopic characterization of Co(7)MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd(4)Cys(11) and Cd(3)Cys(9) clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd(7)MT-4 is cooperative and sequential, with the Cd(4)Cys(11) cluster being formed first, and that a distinct single-metal nucleation intermediate Cd(1)MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd(7)MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd(7)MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.     

Genetic structure of the Eurasian lynx population in north-eastern Poland and the Baltic states

We analyzed the genotypes of Eurasian lynx (Lynx lynx) from three populations in the westernmost part of the species main range. One population was situated at the distribution edge (NE Poland) and the two other (Latvia and Estonia) were located within the main, contiguous range of the species. The aim was to determine if the genetic composition varied among these populations and if there was evidence of isolation among them. Based on microsatellite allele frequencies, we found the allelic richness in Polish lynx to be lower than that in lynx from Latvia and Estonia. We also found significant differentiation among the lynx populations, with the NE Poland population forming a distinct genetic group relative to the two other populations (R _ST = 0.15 and 0.22, P < 0.0001). We suggest that genetic differentiation among lynx populations is the result of habitat insularisation that limits gene flow. This finding emphasizes the necessity to consider the lynx genetic differentiation in conservation planning of this species in Poland.