Are pollen records from small sites appropriate for REVEALS model-based quantitative reconstructions of past regional vegetation? An empirical test in southern Sweden

In this paper we test the performance of the Regional Estimates of VEgetation Abundance from Large Sites (REVEALS) model using pollen records from multiple small sites. We use Holocene pollen records from large and small sites in southern Sweden to identify what is/are the most significant variable(s) affecting the REVEALS-based reconstructions, i.e. type of site (lakes and/or bogs), number of sites, site size, site location in relation to vegetation zones, and/or distance between small sites and large sites. To achieve this objective we grouped the small sites according to (i) the two major modern vegetation zones of the study region, and (ii) the distance between the small sites and large lakes, i.e. small sites within 50, 100, 150, or 200 km of the large lakes. The REVEALS-based reconstructions were performed using 24 pollen taxa. Redundancy analysis was performed on the results from all REVEALS-model runs using the groups within (i) and (ii) separately, and on the results from all runs using the groups within (ii) together. The explanatory power and significance of the variables were identified using forward selection and Monte Carlo permutation tests. The results show that (a) although the REVEALS model was designed for pollen data from large lakes, it also performs well with pollen data from multiple small sites in reconstructing the percentage cover of groups of plant taxa (e.g. open land taxa, summer-green trees, evergreen trees) or individual plant taxa; however, in the case of this study area, the reconstruction of the percentage cover of Calluna vulgaris, Cyperaceae, and Betula may be problematic when using small bogs; (b) standard errors of multiple small-site REVEALS estimates will generally be larger than those obtained using pollen records from large lakes, and they will decrease with increasing size of pollen counts and increasing number of small sites; (c) small lakes are better to use than small bogs if the total number of small sites is low; and (d) the size of small sites and the distance between them do not play a major role, but the distance between the small sites and landscape/vegetation boundaries is a determinant factor for the accuracy of the vegetation reconstructions.     

Synthesis and SAR of succinamide peptidomimetic inhibitors of cathepsin S

Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.     

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.     

High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.     

Insights into the quality of DnaA boxes and their cooperativity

Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replication-inactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperativity between the DnaA boxes, and to study in vivo the in vitro-defined 9mer DnaA box consensus sequence (TT(A)/(T)TNCACA). The quality and cooperativity of the DnaA boxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box in the promoter-distal position, whereas a promoter-proximal DnaA box with the sequence TTATCCACA titrated DnaA protein and caused significant repression of the mioC promoter without a promoter-distal DnaA box. The quality of the eight different consensus DnaA boxes located in the promoter-proximal position was determined: TTATCCACA had the highest affinity for DnaA protein. In the third position, A was better than T, and the four possibilities in the fifth position could be ranked as C >A >or=G >T. Parallel in vitro experiments using a purified DNA-binding domain of DnaA protein gave the same ranking of the binding affinities of the eight DnaA boxes.     

Allele-specific suppression of dnaA(Ts) mutations by rpoB mutations in Escherichia coli

Summary Extragenic suppressor mutations for dnaA(Ts) mutations mapping in the rpoB gene (-subunit of RNA polymerase) were isolated by selection of spontaneous rifampicin resistant mutants and screening for temperature resistance. Six rpoB mutations were analysed for suppression of 12 different dnaA(Ts) mutations. The analysis showed that all dnaA(Ts) mutations could be suppressed by some rpoB mutation. All six rpoB mutations showed allele specificity when tested for suppression of 12 dnaA (Ts) mutant strains. The allele specificity was found to correlate with the map position of the dnaA (Ts) alleles.     

Biohydrogenation of unsaturated fatty acids. Hydrogenation by cell-free preparations of Butyrivibrio fibrisolvens.

Hydrogenation of cis-9,trans-11-octadecadienoic acid to yield trans-11-octadecenoic acid by cell-free preparations of Butyrivibrio fibrisolvens has been obtained under strictly anaerobic conditions. Reduced methyl viologen, NADH, and an endogenous electron donor each can serve as a reductant. Inhibition studies and gel filtration patterns reveal the presence of at least two hydrogenation systems, one of which is coupled through a flavin, possibly FMN. Although the enzymes comprising the biohydrogenation pathway, the fatty acid reductases and linoleic acid isomerase, are part of the bacterial membrane, they do not appear to be constituted as a multienzyme complex.     

The role of nitric oxide in the cardiac effects of hydrogen peroxide

Oxidative stress mediated by hydrogen peroxide (H₂O₂) increases coronary flow (CF) in Langendorff-perfused rat hearts. We investigated the possible role of nitric oxide (NO) in H₂O₂-induced vasolidation. A dose-response study was conducted to find a concentration of H₂O₂ which increased CF without influencing left ventricular developed (LVDP) or end-diastolic (LVEDP) pressures. 80 (n = 10),100 (n = 7), 120 (n = 7),140 (n = 7),160 (n = 7), and 180 (n = 10) M H₂O₂ was infused for 10 min, followed by recovery for 50 min. 80 M H₂O₂ increased CF to a maximum of 143 ± 4 (mean ± S.E.M) percent of initial value after 15 min observation (p < 0.001 compared to buffer only), with no effect on LVDP or LVEDP. Another series of hearts were perfused with N-nitro-L-Arginine methylester (L-NAME, 1 M), methylene blue (MB, 50 M), or haemoglobin (Hb, 10 M), without (n = 7 in each) or with (n = 10 in each) 80 M H₂O₂ for 10 min. L-NAME, MB, and Hb alone increased CF, but attenuated the H₂O₂-induced increase of CF. LVDP was depressed when L-NAME, MB, or Hb were given in conjunction with 80 M H₂O₂. In summary, H₂O₂ concentration-dependently increased LVEDP and depressed LVDP. The H₂O₂-induced increase of CF was independent of concentration. Inhibition of NO synthesis, action, or soluble guanylate cyclase attenuated the H₂O₂-induced increase of CF, and depressed LVDP when given together with H₂O₂. H₂O₂ induces a NO-dependent vasodilation, and inhibition of NO is detrimental to left ventricular function after H₂O₂-mediated oxidative stress.