Impact of Plant Species and Site on Rhizosphere-Associated Fungi Antagonistic to Verticillium dahliae Kleb.

Fungi with antagonistic activity toward plant pathogens play an essential role in plant growth and health. To analyze the effects of the plant species and the site on the abundance and composition of fungi with antagonistic activity toward Verticillium dahliae, fungi were isolated from oilseed rape and strawberry rhizosphere and bulk soil from three different locations in Germany over two growing seasons. A total of 4,320 microfungi screened for in vitro antagonism toward Verticillium resulted in 911 active isolates. This high proportion of fungi antagonistic toward the pathogen V. dahliae was found for bulk and rhizosphere soil at all sites. A plant- and site-dependent specificity of the composition of antagonistic morphotypes and their genotypic diversity was found. The strawberry rhizosphere was characterized by preferential occurrence of Penicillium and Paecilomyces isolates and low numbers of morphotypes (n = 31) and species (n = 13), while Monographella isolates were most frequently obtained from the rhizosphere of oilseed rape, for which higher numbers of morphotypes (n = 41) and species (n = 17) were found. Trichoderma strains displayed high diversity in all soils, but a high degree of plant specificity was shown by BOX-PCR fingerprints. The diversity of rhizosphere-associated antagonists was lower than that of antagonists in bulk soil, suggesting that some fungi were specifically enriched in each rhizosphere. A broad spectrum of new Verticillium antagonists was identified, and the implications of the data for biocontrol applications are discussed.     

Über das Riechvermögen und den bau des Geruchs-Organs von Triton Vulgaris vor und Nach der Metamorphose

Ohne ZusammenfassungDie Arbeit wurde von der Philos. Fakultät Greifswald als Dissertation angenommen.     

Cardiac troponin C-L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C

We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium-binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt-Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine-glutamine exchange at position 29 in the nonfunctional calcium-binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N-terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107-112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A-dependent phosphorylation of cTnI at serine 22 and serine 23 in wild-type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+-free and Ca2+-saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+-dependent actomyosin subfragment 1 (actoS1)-ATPase activity and the sliding velocity of thin filaments. The maximum actoS1-ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A-dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1-ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A-dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.     

Single neonatal treatment with beta-endorphin (hormonal imprinting) extremely enhances nocistatin level of cerebrospinal fluid in adult rats

In earlier experiments endorphin treatment of newborn rats caused the decrease of brain serotonin content, increasing aggressivity, enhanced sexual activity of females and changes in the binding capacity of uterine estrogen receptors at adult age, however nociceptin content of the cerebrospinal fluid was not changed. In the present experiment neonatal treatment of male and female rats was done with a single dose of 3 microg beta-endorphin and in five months old rats the level of nociceptin antagonist nocistatin was determined by radioimmunoassay in the cerebrospinal fluid. In both genders the amount of nocistatin was one magnitude higher in the endorphin treated groups. There was also a significant difference between the male and female nocistatin level in the treated and non-treated groups alike, with the advantage of females. The results call attention to the possibility of influencing pain-tolerance for life, by the pain-provoked endorphin levels during delivery.     

Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells

One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fc part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG₁ molecule. In the present study, culture supernatant of a stable clone was collected and purified by affinity chromatography prior characterization. We emphasized product quality aspects regarding the fusion protein itself and in addition, post-translational characterization of the subunits in comparison to human antibodies and rhEpo. However, overproduction of recombinant proteins in mammalian cells is well established, analysis of product quality of complex products for different purposes, such as product specification, purification issues, batch to batch consistency and therapeutical consequences, is required. Besides product quantification by ELISA, N-acetylneuraminic acid quantification in microtiterplates, quantitative isoform pattern and entire glycan profiling was performed. By using these techniques for the characterization of the recombinant human Epo-Fc (rhEpo-Fc) molecule itself and furthermore, for the separate characterization of both subunits, we could clearly show that no significant differences in the core glycan structures compared to rhEpo and human antibody N-glycans were found. The direct comparison with other rhEpo-Fc fusion proteins failed, because no appropriate data were found in the literature.