Root growth inhibition of the holorganic layer of moder humus under spruce (Picae abies KARST.) and beech (Fagus Silvatica L.)

A test of root growth inhibition of spruce and beech roots, according to Lynch's procedure (1977), shows the inhibitory effects of soil solution extracted from the holorganic layers (Of₂-Oh) under beech and spruce. Molecular gel filtration of soil solutions shows that the molecular weights vary over a wide range, from less than 100 to over 40,000 daltons. Chemical analysis, using CGC, HPLC and sometimes MS shows only negligible concentrations of simple aliphatic (C₁-C₅) and aromatic acids in the free state. Using the fraction scheme of Forsyth (1977) and the carbazole procedure, it is shown that uronic acids represent only a small percentage of the carboxylic acids, and have no inhibitory effects on root growth. By analogy with results of other authors, the presence of polycarboxylic acids in the soil solution are considered to be the main cause of root growth inhibition.     

Purification and characterization of chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica)

1. A chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica) was purified by acid extraction, salt fractionation and chromatographic separation on CM-cellulose and Sephadex G-100, and gave a single protein band on SDS-PAGE. 2. Quail chymotrypsinogen had a mol. wt of 26,100 calculated from amino acid composition data, an isoelectric point of 7.68, a Km of 3.1 mM and K0 of 40.7 sec-1 for tyrosine ester substrate. 3. The activated chymotrypsinogen of quail had a maximum activity at pH 7.0-8.0 and at 45 degrees C, and was stable at pH 4.0-6.0 below 55 degrees C. 4. Comparison of quail and bovine chymotrypsinogens indicates that the activities of the enzymes from quail and bovine are more constant than their physical characteristics.     

Association between COMT SNP variation and timidity in Golden and Labrador Retrievers

Timidity in dogs is a trait with high heritability. However, the relevant genetic factors and markers associated with this condition are largely unknown. The function of the catechol‐O‐methyl transferase (COMT) gene has been found to be associated with human fearful or anxious emotions, and the COMT:p.Val158Met polymorphism locus is significantly related to anxious behavior. In the present study, the correlation between timidity and four single nucleotide polymorphism (SNP) variations (C.‐1666C>G c.39A>G, c.216G>A, c.482G>A) of the COMT gene was investigated in dogs. The evaluation was based on the dog courage assessment assay and a genotype and haplotype analysis in Labrador Retrievers (LR) and Golden Retrievers (GR). The principal components analysis factor structure of the courage phenotype was invariant between LR and GR. Sex, breed and age had no statistically significant effect on the timidity of the dogs. All SNP loci detected were in Hardy–Weinberg equilibrium. The c.39A>G locus was removed in the subsequent association analysis due to the significant difference between LR and GR in genotype distributions. Intriguingly, the genotypes and haplotypes of the COMT gene were significantly and highly correlated with the timidity of LR and GR. The A alleles of the COMT:c.216G>A and c.482G>A loci appeared to play a dominant role in the timid behavior of the dogs. This result supports and broadens the warrior/worrier hypothesis and will have important implications for the understanding of the evolution of temperament in dogs. Additionally, the results provide predictive genetic markers for temperament in dogs.     

Interaction of mutant influenza virus hemagglutinin fusion peptides with lipid bilayers: probing the role of hydrophobic residue size in the central region of the fusion peptide.

The amino-terminal region of the membrane-anchored subunit of influenza virus hemagglutinin, the fusion peptide, is crucial for membrane fusion of this virus. The peptide is extruded from the interior of the protein and inserted into the lipid bilayer of the target membrane upon induction of a conformational change in the protein by low pH. Although the effects of several mutations in this region on the fusion behavior and the biophysical properties of the corresponding peptides have been studied, the structural requirements for an active fusion peptide have still not been defined. To probe the sensitivity of the fusion peptide structure and function to small hydrophobic perturbations in the middle of the hydrophobic region, we have individually replaced the alanine residues in positions 5 and 7 with smaller (glycine) or bulkier (valine) hydrophobic residues and measured the extent of fusion mediated by these hemagglutinin constructs as well as some biophysical properties of the corresponding synthetic peptides in lipid bilayers. We find that position 5 tolerates a smaller and position 7 a larger hydrophobic side chain. All peptides contained segments of alpha-helical (33-45%) and beta-strand (13-16%) conformation as determined by CD and ATR-FTIR spectroscopy. The order parameters of the peptide helices and the lipid hydrocarbon chains were determined from measurements of the dichroism of the respective infrared absorption bands. Order parameters in the range of 0.0-0.6 were found for the helices of these peptides, which indicate that these peptides are most likely aligned with their alpha-helices at oblique angles to the membrane normal. Some (mostly fusogenic) peptides induced significant increases of the order parameter of the lipid hydrocarbon chains, suggesting that the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface.     

Soluble recombinant influenza vaccines.

Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the neuraminidase gene to a tetramerizing leucine zipper sequence; the resulting product was enzymatically active, tetrameric neuraminidase. The protective immunity induced by this engineered neuraminidase, however, remained fairly strain-specific. A third influenza A virus protein, the M2 protein, has only 23 amino acids exposed on the outer membrane surface. This extracellular part, M2e, has been remarkably conserved in all human influenza A strains since 1933. By fusing the M2e sequence to hepatitis B virus core protein, we could obtain highly immunogenic particles that induced complete, strain-independent, long-lasting protection in mice against a lethal viral challenge. Native M2 is a tetrameric protein and this conformation of the M2e part can also be mimicked by fusing this sequence to a tetramerizing leucine zipper. The potential of the resulting protein as a vaccine candidate remains to be evaluated.     

Fluorinated non-imidazole histamine H3 receptor antagonists

Fluorine substituents have become a widespread and important component in drug design and development. Here, the synthesis of fluorine containing compounds and some corresponding precursor molecules are presented for potential isotope labelling as well as their data obtained with in vitro and in vivo screenings. The compounds vary in the basic centres (piperidine or pyrrolidine) and are fluoro substituted in different positions of the basic alicyclic moiety. Pharmacological evaluation resulted in ligands with high affinities at hH₃ receptor in the nanomolar and subnanomolar concentration range and some with high antagonist in vivo potencies.