Spectroscopic studies of perturbed T1 Cu sites in the multicopper oxidases Saccharomyces cerevisiae Fet3p and Rhus vernicifera laccase: allosteric coupling between the T1 and trinuclear Cu sites

The multicopper oxidases catalyze the 4e- reduction of O2 to H2O coupled to the 1e- oxidation of 4 equiv of substrate. This activity requires four Cu atoms, including T1, T2, and coupled binuclear T3 sites. The T2 and T3 sites form a trinuclear cluster (TNC) where O2 is reduced. The T1 is coupled to the TNC through a T1-Cys-His-T3 electron transfer (ET) pathway. In this study the two T3 Cu coordinating His residues which lie in this pathway in Fet3 have been mutated, H483Q, H483C, H485Q, and H485C, to study how perturbation at the TNC impacts the T1 Cu site. Spectroscopic methods, in particular resonance Raman (rR), show that the change from His to Gln to Cys increases the covalency of the T1 Cu-S Cys bond and decreases its redox potential. This study of T1-TNC interactions is then extended to Rhus vernicifera laccase where a number of well-defined species including the catalytically relevant native intermediate (NI) can be trapped for spectroscopic study. The T1 Cu-S covalency and potential do not change in these species relative to resting oxidized enzyme, but interestingly the differences in the structure of the TNC in these species do lead to changes in the T1 Cu rR spectrum. This helps to confirm that vibrations in the cysteine side chain of the T1 Cu site and the protein backbone couple to the Cu-S vibration. These changes in the side chain and backbone provide a possible mechanism for regulating intramolecular T1 to TNC ET in NI and partially reduced enzyme forms for efficient turnover.     

Changes in the fatty acid unsaturation after hardening in wheat chromosome substitution lines with different cold tolerance

Two wheat (Triticum aestivum L.) varieties, Cheyenne (Ch, winter wheat with excellent frost tolerance) and Chinese Spring (CS, spring wheat with weak frost tolerance), and chromosome substitution lines (CS/Ch 5A, CS/Ch 5D, CS/Ch 7A) created from Cheyenne and Chinese Spring were used to study the effect of chromosome substitutions on the membrane lipid composition in the leaves and crowns before and after cold hardening. The percentage of fatty acid unsaturation in phosphatidylethanolamine was greater in the crown of hardened Cheyenne than in Chinese Spring. The value of CS/Ch 5A was similar to Cheyenne and that of CS/Ch 5D to Chinese Spring, while the value of CS/Ch 7A was in between those of Cheyenne and Chinese Spring. A smaller difference was found between the unsaturation level in the phosphatidylcholine from Cheyenne and Chinese Spring after hardening, while the value obtained for the substitution line CS/Ch 7A was similar to Cheyenne. The percentage decrease in thetrans-Δ³-hexadecenoic acid content was found to be correlated with the frost tolerance of the wheat genotypes.     

A synthetic RNA-mediated evolution system in yeast

Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in baker''s yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.     

In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket.

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.     

Bonobo (Pan paniscus) Spatial Memory and Communication in a 20-hectare Forest

We used an artificial language as a tool for the study of spatial memory organization in a young Pan paniscus. In the first experiment, we showed the bonobo a road sign just outside its indoor sleeping area. The sign indicated, by means of arbitrarily designated geometrical shapes (lexigrams), where food was hidden. Only 2 of the 15 locations were visible from the sign. Distances ranged up to 170 m from the sign. In 99 of 127 test trials the bonobo went directly to the designated location on its first move. In a second experiment, we presented the road sign at varied points in the woods rather than at the original fixed place. In these trials the goal was a preferred toy. The bonobo's human companions were never told the location of the goal and distances were up to 650 m. In all 12 trials the bonobo led its companions to the designated place via an efficient path. The bonobo appeared to be able to move, based on the information provided by a lexigram, from almost any arbitrary starting location in its 20-ha environment to any one of the numerous goal locations.     

Wood plant communities of the Bukovské vrchy Hills,NE Slovakia

Three shrub associations and 11 forest associations with 8 subassociations were found in the Bukovské vrchy hills. Three associations are described as new.     

Probleme der Inkretwirkungen

Ohne Zusammenfassung     

Staining Aspirated Human Bone Marrow with Domestic Wright Stain

The method employs the domestic Wright stain for the staining of aspirated human bone marrow. Freshly distilled water, pH 6.0 to 6.4, is used. Wright stain, 0.5 cc, is placed upon the air-dried preparation and permitted to act for two minutes. The stain is then diluted with 2 cc. distilled water and permitted to act for from 5 to 10 minutes. After washing off the stain with distilled water, the preparation is placed into a decolorizer (acetone 0.5 cc, pure methyl alcohol 5.0 cc, and 100 cc. distilled water, pH 6.0 to 6.4) for differentiation from 1 to 5 seconds, rinsed, washed under running water and permitted to air-dry. A well stained and differentiated preparation shows the “Romanovsky effect”, and the sharpness of minute structures obtained compares favorably with control preparations stained with German dyes.

The bone marrow should be prepared as described. The Wright stain marketed by the National Aniline and Chemical Co., N. Y. was found to be reliable as regards staining quality of registered batches. One photomicrograph, showing bone marrow cells from pernicious anemia, is included.