Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins

AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.     

Associations of HLA-A, -B and -DRB1 Types with Oral Diseases in Swiss Adults

Human leukocyte antigens (HLA) are crucial components of host defense against microbial challenge but the associations of HLA types with oral infectious diseases have not been studied in detail. This prospective cross-sectional study examined associations of HLA-A, -B and -DRB1 types with common oral diseases in a healthy Swiss adult population. 257 subjects (107 m, 150 f, mean age: 43.5 yr; range: 21–58 yr) with known HLA-A, -B and -DRB1 profiles and comprehensive medical records were included. A thorough anamnesis was followed by oral examinations including saliva flow measurements, the DMFT score for cariological status, complete periodontal status with plaque and bleeding indexes as well as assessment of mucosal alterations and temporomandibular dysfunction (TMD). Student’s t-test and Pearson chi-square test were utilized to compare the oral diseases between HLA positive and negative subjects. Bonferroni correction for multiple comparisons was used and P_Bonf<0.05 was considered statistically significant. HLA types -B15 (P_Bonf = 0.002), -B51 (P_Bonf = 0.02) and -DRB1*12 (P_Bonf = 0.02) were associated with less periodontal disease manifestations. HLA-A32 had a positive association with TMD dysfunction (P_Bonf = 0.012). No other statistically significant associations were observed. In conclusion, HLA types may contribute to the development of oral diseases in generally healthy Caucasian adults.     

Evolutionary Genomics of Structural Variation in Asian Rice (Oryza sativa) Domestication

Structural variants (SVs) are a largely unstudied feature of plant genome evolution, despite the fact that SVs contribute substantially to phenotypes. In this study, we discovered SVs across a population sample of 347 high-coverage, resequenced genomes of Asian rice (Oryza sativa) and its wild ancestor (O. rufipogon). In addition to this short-read data set, we also inferred SVs from whole-genome assemblies and long-read data. Comparisons among data sets revealed different features of genome variability. For example, genome alignment identified a large (∼4.3 Mb) inversion in indica rice varieties relative to japonica varieties, and long-read analyses suggest that ∼9% of genes from the outgroup (O. longistaminata) are hemizygous. We focused, however, on the resequencing sample to investigate the population genomics of SVs. Clustering analyses with SVs recapitulated the rice cultivar groups that were also inferred from SNPs. However, the site-frequency spectrum of each SV type—which included inversions, duplications, deletions, translocations, and mobile element insertions—was skewed toward lower frequency variants than synonymous SNPs, suggesting that SVs may be predominantly deleterious. Among transposable elements, SINE and mariner insertions were found at especially low frequency. We also used SVs to study domestication by contrasting between rice and O. rufipogon. Cultivated genomes contained ∼25% more derived SVs and mobile element insertions than O. rufipogon, indicating that SVs contribute to the cost of domestication in rice. Peaks of SV divergence were enriched for known domestication genes, but we also detected hundreds of genes gained and lost during domestication, some of which were enriched for traits of agronomic interest.     

Role of clozapine in the occurrence of chromosomal abnormalities in human bone-marrow cells in vivo and in cultured lymphocytes in vitro

Summary Bone-marrow chromosomes were examined from 38 mentally and physically retarded and two psychiatric patients who were being treated with a variety of neuropharmacologic drugs. Twenty of these patients used clozapine (Leponex^®). The clastogenic effects of clozapine in vitro were studied in the lymphocyte cultures of three patients-one free of hematologic disease and two who 6 months earlier had had agranulocytosis attributed to the use of clozapine. The mean frequency of cytogenetic abnormalities in the bonemarrow cells of patients who used clozapine was significantly increased (P> 0.05). The two patients who had had agranulocytosis had a greater frequency of cytogenetic abnormalities in their cultured lymphocytes in vivo and in vitro than the patient free of hematologic disease. A clone with a 13/14 chromosome translocation was detected in one of the patients. As all patients received a number of drugs during the in vivo and in vitro studies no definite conclusions could be drawn regarding the role played by clozapine in the occurrence of chromosomal abnormalities.     

Impact of structural changes in wood‐using industries on net carbon emissions in Finland

Forests and forest industries can contribute to climate change mitigation by sequestering carbon from the atmosphere, by storing it in biomass, and by fabricating products that substitute more greenhouse gas emission intensive materials and energy. The objectives of the study are to specify alternative scenarios for the diversification of wood product markets and to determine how an increasingly diversified market structure could impact the net carbon emissions (NCEs) of forestry in Finland. The NCEs of the Finnish forest sector were modelled for the period 2016–2056 by using a forest management simulation and optimization model for the standing forests and soil and separate models for product carbon storage and substitution impacts. The annual harvest was fixed at approximately 70 Mm³, which was close to the level of roundwood removals for industry and energy in 2016. The results show that the substitution benefits for a reference scenario with the 2016 market structure account for 9.6 Mt C (35.2 Mt CO₂ equivalent [CO₂ eq]) in 2056, which could be further increased by 7.1 Mt C (26 Mt CO₂ eq) by altering the market structure. As a key outcome, increasing the use of by‐products for textiles and wood–plastic composites in place of kraft pulp and biofuel implies greater overall substitution credits compared to increasing the level of log harvest for construction.     

PKCepsilon-mediated phosphorylation of vimentin controls integrin recycling and motility

PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.     

Characterisation of a secondary alcohol dehydrogenase from Xanthomonas campestris DSM 3586

The chromosomal locus NP_636946 of Xanthomonas campestris DSM 3586 (ATCC 33913) which was earlier presumed to encode a quinoprotein glucose dehydrogenase has been cloned, expressed in Escherichia coli and the recombinant enzyme has been characterised. It was found to have no glucose dehydrogenase activity but to be active on many different polyols and diols, aliphatic alcohols, certain aldonic acids and amino-sugars. The product of d-gluconic acid oxidation was 5-keto-d-gluconic acid. The enzyme differs from polyol/gluconate dehydrogenases found in Gluconobacter by its single-chain architecture, different substrate specificity and much higher (20- to 30-fold) expression level in E.coli.     

Birch PR-10c interacts with several biologically important ligands

PR-10c is a unique member of PR-10 proteins in birch, since it is the only one known to be post-translationally modified by glutathione and is not constitutively expressed in pollen. Both reduced and S-glutathiolated forms of PR-10c show low ribonuclease activity. However, the major function of the protein is apparently not yet resolved. Our protein-ligand interaction studies with saturation transfer difference (STD) NMR revealed that PR-10c interacts with several biologically important molecules, including cytokinin, flavonoid glycosides, sterols and emodin. Competition study with deoxycholate and kinetin revealed no statistically significant binding interference, indicating that these ligands have different binding sites in PR-10c. Ligand docking studies with a molecular model of PR-10c support the STD NMR results of ligand binding and binding epitopes, suggesting that there are three potential binding sites in PR-10c: two in the hydrophobic cavity and one in the glycine-rich loop. Our docking calculations suggested that only kinetin interacts with the glycine-rich loop, the binding occurring through its adenine moiety. Clear ligand specificity could be observed in the binding of nucleotide derivatives. S-glutathiolation of PR-10c did not affect kinetin binding. The present results suggest that birch PR-10c is a multifunctional protein, which has diverse roles in plant stress responses.