Effects of Recreational Cabins,Trails and Their Removal for Restoration of Reindeer Winter Ranges

Conservation efforts have secured the partial recovery of Europe's wild reindeer, although only in 24 separate fragments of their original range, now separated by resorts and roads. Full recovery of the original range will require restoration of migration routes across developed or disturbed areas. We analyzed distribution of around 3500 Rangifer tarandus tarandus (reindeer) during winters (1984–2005) in relation to 10 alpine resorts and prior to and following relocation of ski trails and cabins in Norway done to restore use of former habitat. Reindeer used areas within 15 km of resorts, which is less than expected based on the availability of habitat, most likely as a result of cross‐country skiing activity surrounding the resorts, limiting their access to other ranges and historic migration corridors. Reindeer abundance declined and mean distance between reindeer groups and resorts increased with increasing resort size. No apparent habituation to resorts was observed during the 20‐year study period. However, when ski trails and an associated tourist cabin were removed to restore access to historic habitat, reindeer moved into the area. No such change in reindeer distribution was observed in the 10 years preceding relocation, or at the other nine resorts where no such experiments were conducted. Regulation of human traffic, relocation of trails, and removal of infrastructure and cabins are apparently effective in restoring access to and use of historic ranges and migration routes. However, restoration of historic migration routes between ranges will likely require the removal of hundreds of recreational cabins in order to become effective.     

Analysis of various conditions in order to measure electromyography of isometric contractions in water and on air

The aim of this study was to verify if there are differences in the amplitude of signals from surface electromyography (EMG) during maximal and submaximal voluntary isometric contraction (MVC and 50% MVC, respectively) under different conditions, in our case, water and air, with and without extra protection (water-resistant tape) on the electrode. The isometric force and muscle activation of the MVC and 50% MVC of the biceps brachial muscle of nine healthy trained men were measured simultaneously, performed in water and on air, with and without protection of the EMG electrode. The multivariate analysis of variance with a post hoc Tukey test was applied to detect significant differences between the levels of muscular force. For the amplitude values of the EMG signal, the Wilcoxon signed rank test was applied to compare all experimental conditions in order to detect a significance of p < 0.05. The values of isometric force were not significantly different among conditions (MVC and 50% MVC). The results showed a significant difference among conditions in the water without extra protection compared to the conditions on air with and without extra protection and in water with extra protection. Reduced EMG amplitude was seen in water without extra protection from 37.04% to 55.81% regarding the other conditions. However, no significant difference was seen among conditions in water with extra protection in relation the conditions on air (with and without extra protection). This study suggest that it is necessary to use a water-resistant tape as an extra protection on the electrode when using EMG underwater, to avoid having a significant decrease in the EMG amplitude underwater and not to suffer interference from the water. There was no significant difference among the recordings of EMG with and without the use of protection on air; therefore, the protection does not influence the recording of EMG amplitude and isometric force on air.     

The current status of the small subunit rRNA phylogeny of the coccidia (Sporozoa)

There is no current comprehensive assessment of the molecular phylogeny of the coccidia, as all recently published papers either deal with subsets of the taxa or sequence data, or provide non-robust analyses. Here, we present a comprehensive and consistent phylogenetic analysis of the available data for the small-subunit ribosomal RNA gene sequence, including a number of taxa not previously studied, based on a Bayesian tree-building analysis and the covariotide model of evolution. The assumptions of the analysis have been rigorously tested, and the benefits and limitations highlighted. Our results provide support for a number of prior conclusions, including the monophyly of the families Sarcocystidae (cyst-forming coccidia) and Eimeriidae (oocyst-forming coccidia), but with bird-host Isospora species in the Eimeriidae and mammal-host species in the Sarcocystidae. However, it is clear that a number of previously reported relationships are dependent on the evolutionary model chosen, such as the placements of Goussia janae, Lankesterella minimia and Caryospora bigenetica. Our results also confirm the monophyly of the subfamilies Toxoplasmatinae and Sarcocystinae, but only some of the previously reported groups within these subfamilies are supported by our analysis. Similarly, only some of the previously reported groups within the Eimeriidae are supported by our analysis, and the genus Eimeria is clearly paraphyletic. There are unambiguous patterns of host-parasite relationship within the coccidia, as most of the well-supported groups have a consistent and restricted range of hosts, with the exception of the Toxoplasmatinae. Furthermore, the previously reported groups for which we found no support all have a diverse range of unrelated hosts, confirming that these are unlikely to be natural groups. The most interesting unaddressed questions may relate to Isospora, which has the fewest available sequences and host-parasite relationships apparently not as straightforward as elsewhere within the suborder.     

Functional properties of the herpes simplex virus type I origin-binding protein are controlled by precise interactions with the activated form of the origin of DNA replication

The herpes simplex virus, type I origin-binding protein, OBP, is a superfamily II DNA helicase encoded by the UL9 gene. OBP binds in a sequence-specific and cooperative way to the viral origin of replication oriS. OBP may unwind partially and introduce a hairpin into the double-stranded origin of replication. The formation of the novel conformation referred to as oriS* also requires the single-stranded DNA-binding protein, ICP8, and ATP hydrolysis. OBP forms a stable complex with oriS*. The hairpin in oriS* provides a site for sequence-specific attachment, and a single-stranded region triggers ATP hydrolysis. Here we use Escherichia coli exonuclease I to map the binding of the C-terminal domain of OBP to the hairpin and the helicase domains to the single-stranded tail. The helicase domains cover a stretch of 23 nucleotides of single-stranded DNA. Using streptavidin-coated magnetic beads, we show that OBP may bind two copies of double-stranded DNA (one biotin-labeled and the other one radioactively labeled) but only one copy of oriS*. It is the length of the single-stranded tail that determines the stoichiometry of OBP.DNA complexes. OBP interacts with the bases of the single-stranded tail, and ATP hydrolysis is triggered by position-specific interactions between OBP and bases in the single-stranded tail of oriS*.     

4-Hydroxyphenylpyruvate dioxygenase: a hybrid density functional study of the catalytic reaction mechanism

Density functional calculations using the B3LYP functional has been used to study the reaction mechanism of 4-hydroxyphenylpyruvate dioxygenase. The first part of the catalytic reaction, dioxygen activation, is found to have the same mechanism as in alpha-ketoglutarate-dependent enzymes; the ternary enzyme-substrate-dioxygen complex is first decarboxylated to the iron(II)-peracid intermediate, followed by heterolytic cleavage of the O-O bond yielding an iron(IV)-oxo species. This highly reactive intermediate attacks the aromatic ring at the C1 position and forms a radical sigma complex, which can either form an arene oxide or undergo a C1-C2 side-chain migration. The arene oxide is found to have no catalytic relevance. The side-chain migration is a two-step process; the carbon-carbon bond cleavage first affords a biradical intermediate, followed by a decay of this species forming the new C-C bond. The ketone intermediate formed by a 1,2 shift of an acetic acid group rearomatizes either at the active site of the enzyme or in solution. The hypothetical oxidation of the aromatic ring at the C2 position was also studied to shed light on the 4-HPPD product specificity. In addition, the benzylic hydroxylation reaction, catalyzed by 4-hydroxymandelate synthase, was also studied. The results are in good agreement with the experimental findings.     

Vesicle membrane interactions of penetratin analogues

Reports on serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides, also denoted protein transduction domains, have demonstrated the need for a reevaluation of the current understanding of peptide-mediated cellular delivery of large, hydrophilic molecules. In a recent study on the internalization in unfixed cells of penetratin and its analogues in which tryptophans are substituted for phenylalanines (Pen2W2F), lysines for arginines (PenArg), and arginines for lysines (PenLys), we revealed large dissimilarities in cell interactions among the peptides [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107]. We here investigated possible correlations with their respective affinities for the lipid membranes of large unilamellar vesicles. The variations found in membrane affinity correlated qualitatively with differences in hydrophobicity among the peptides but were by far too small to account for the striking differences in cell membrane binding. Interestingly, we found that the inclusion of a small fraction of lipids conjugated to poly(ethylene glycol) (PEG) in the vesicles both stabilized the vesicle dispersion against peptide-induced aggregation and, furthermore, enhanced the binding of the peptides to the membrane. By use of PEG-conjugated lipids, it could be shown that vesicle aggregation drives an alpha-helix to beta-sheet conformational transition for these peptides. A similar transition was discovered at submicellar concentrations of sodium dodecyl sulfate in aqueous solution for all peptides except PenLys. Finally, significant changes of the contributions to CD spectra from aromatic residues due to their insertion into the membrane were observed.     

Insulin induces SOCS-6 expression and its binding to the p85 monomer of phosphoinositide 3-kinase, resulting in improvement in glucose metabolism

The suppressors of cytokine signaling (SOCS) family is thought to act largely as a negative regulator of signaling by cytokines and some growth factors. Surprisingly, the SOCS-6 transgenics had no significant defects in the cytokine signaling and hematopoietic system but displayed significant improvements in glucose metabolism. Insulin stimulation of Akt/protein kinase B was also potentiated. Biochemical analysis showed that, after insulin stimulation, SOCS-6 interacted with the monomeric p85 subunit of class-Ia phosphoinositide (PI) 3-kinase but not with p85/p110 dimers. Furthermore, SOCS-6 expression is transiently increased by serum and insulin in normal fibroblasts. However, both the mRNA and protein of SOCS-6 were rapidly degraded after induction by insulin. The degradation of the SOCS-6 protein was partially inhibited by a proteasome inhibitor, suggesting a proteasome-mediated degradation mechanism. In contrast, SOCS-6-associated p85 was not degraded and could be recruited to the newly synthesized SOCS-6 molecules in the presence of insulin, suggesting that SOCS-6 expression and its interaction with p85, but not the degradation, is regulated by insulin. The phenotype of SOCS-6 transgenic mice bears a striking resemblance to p85 knock-out mouse models in which glucose metabolism stimulated by insulin is significantly improved despite reduced activation of PI 3-kinase. This suggests that monomeric p85 might play a physiologically important role in attenuating signaling through PI 3-kinase-dependent pathways in unstimulated cells. Therefore, our results indicate that SOCS-6 may provide a dynamically regulated mechanism by which insulin can transiently overcome the negative effects that p85 monomers have on signaling via PI 3-kinase-dependent signaling pathways.     

Osmotic properties of poly(ethylene glycols): quantitative features of brush and bulk scaling laws

From glycosylated cell surfaces to sterically stabilized liposomes, polymers attached to membranes attract biological and therapeutic interest. Can the scaling laws of polymer "brushes" describe the physical properties of these coats? We delineate conditions where the Alexander-de Gennes theory of polymer brushes successfully fits the intermembrane distance versus applied osmotic stress data of Kenworthy et al. for poly(ethylene glycol)-grafted multilamellar liposomes. We establish that the polymer density and size in the brush must be high enough that, in a bulk solution of equivalent monomer density, the polymer osmotic pressure is independent of polymer molecular weight (the des Cloizeaux semidilute regime of bulk polymer solutions). The condition that attached polymers behave as semidilute bulk solutions offers a rigorous criterion for brush scaling-law behavior. There is a deep connection between the behaviors of semidilute polymer solutions in bulk and polymers grafted to a surface at a density such that neighbors pack to form a uniform brush. In this regime, two-parameter unconstrained fits of the Alexander-de Gennes brush scaling laws to the Kenworthy et al. data yield effective monomer lengths of 3.3-3.6 A, which agree with structural predictions. The fitted distances between grafting sites are larger than expected from the nominal mole fraction of poly(ethylene glycol)-lipids; the chains apparently saturate the surface. Osmotic stress measurements can be used to estimate the actual densities of membrane-grafted polymers.     

Structure of the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii and its relation to other homotrimeric dUTPases

The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction.