Polymorphisms and variants in the prion protein sequence of European moose (Alces alces), reindeer (Rangifer tarandus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in Scandinavia

The prion protein (PrP) sequence of European moose, reindeer, roe deer and fallow deer in Scandinavia has high homology to the PrP sequence of North American cervids. Variants in the European moose PrP sequence were found at amino acid position 109 as K or Q. The 109Q variant is unique in the PrP sequence of vertebrates. During the 1980s a wasting syndrome in Swedish moose, Moose Wasting Syndrome (MWS), was described. SNP analysis demonstrated a difference in the observed genotype proportions of the heterozygous Q/K and homozygous Q/Q variants in the MWS animals compared with the healthy animals. In MWS moose the allele frequencies for 109K and 109Q were 0.73 and 0.27, respectively, and for healthy animals 0.69 and 0.31. Both alleles were seen as heterozygotes and homozygotes. In reindeer, PrP sequence variation was demonstrated at codon 176 as D or N and codon 225 as S or Y. The PrP sequences in roe deer and fallow deer were identical with published GenBank sequences.     

Molecular mechanisms of Streptococcus dysgalactiae subsp equisimilis enabling intravascular persistence

Streptococcus dysgalactiae subsp. equisimilis (SDSE) can cause recurrent bacteremic infection. We have characterized novel virulence properties of an SDSE isolate of type stG485.0 that caused severe sepsis three times in a patient despite that he had opsonizing antibodies to the isolate. An infected aortic aneurysm was suspected to be the focus for the persisting bacteria. For the first time we show that this SDSE isolate, as well as other invasive SDSE isolates, aggregate human platelets and efficiently internalize into human endothelial cells. These properties may aid SDSE to persist and could explain the tendency of SDSE to cause recurrent bacteremia.     

Multilocus sequence typing and repetitive-sequence-based PCR (DiversiLab) for molecular epidemiological characterization of Propionibacterium acnes isolates of heterogeneous origin

Propionibacterium acnes is a gram-positive bacillus predominantly found on the skin. Although it is considered an opportunistic pathogen it is also been associated with severe infections. Some specific P. acnes subtypes are hypothesized to be more prone to cause infection than others. Thus, the aim of the present study was to investigate the ability to discriminate between P. acnes isolates of a refined multilocus sequence typing (MLST) method and a genotyping method, DiversiLab, based on repetitive-sequence-PCR technology. The MLST and DiversiLab analysis were performed on 29 P. acnes isolates of diverse origins; orthopedic implant infections, deep infections following cardiothoracic surgery, skin, and isolates from perioperative tissue samples from prostate cancer. Subtyping was based on recA, tly, and Tc12S sequences. The MLST analysis identified 23 sequence types and displayed a superior ability to discriminate P. acnes isolates compared to DiversiLab and the subtyping. The highest discriminatory index was found when using seven genes. DiversiLab was better able to differentiate the isolates compared to the MLST clonal complexes of sequence types. Our results suggest that DiversiLab can be useful as a rapid typing tool for initial discrimination of P. acnes isolates. When better discrimination is required, such as for investigations of the heterogeneity of P. acnes isolates and its involvement in different pathogenic processes, the present MLST protocol is valuable.     

Specific leaf area as a superior predictor of changes in field layer abundance during forest succession

Question : How accurately can a suite of suggested functional traits predict plant species response to succession from semi‐open woodland to closed deciduous canopy forest? Location : Southeastern Sweden. Methods : Abundance of 46 field‐layer plant species in a temperate deciduous forest, measured as frequency of occupied plots, was estimated in 1961, 1970 and 2003. Abundance change over time across species was tested for correlations with functional traits and literature information on habitat preference. Results : Increase in abundance was positively correlated with specific leaf area (SLA), weakly negatively correlated with seed mass and not significantly correlated with plant height or start, peak and length of the flowering period. Change in abundance was correlated with the Ellenberg light indicator value, whereas no correlations were found with Ellenberg values for nitrogen, calcium and moisture, or forest preference according to the literature. Conclusions : SLA was a better predictor of how field layer plants responded to succession from semi‐open woodland to closed canopy forest than empirically‐derived measures of habitat preference. The same holds for SLA in relation to seed size, indicating that interactions in the established life‐cycle phase are more important than the recruitment phase for species response to succession.     

Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.     

On the chemical basis of the Lowry protein determination

The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.