The potential of antagonistic fungi for control of Fusarium graminearum and Fusarium crookwellense varies depending on the experimental approach.

AIMS: To investigate the potential of fungal antagonists to control Fusarium head blight (FHB) causing pathogens (Fusarium graminearum and F. crookwellense) with two different experimental approaches. METHODS AND RESULTS: Using two in vitro tests, Clonostachys rosea, Cladosporium cladosporioides and 10 Trichoderma strains were screened. In a co-culture assay, all Trichoderma strains significantly reduced the colony area of F. graminearum and F. crookwellense by 45-93%, whereas C. rosea and C. cladosporioides were not effective. In another assay, all antagonists from a chosen subset reduced the number of perithecia and ascospores on wheat straw by 88-100% when inoculated before the pathogen. Only C. rosea, a weak antagonist in the co-culture assay, was effective when inoculated after the pathogen, reducing perithecia and ascospore production by 73 and 100%, respectively. CONCLUSIONS: For screening antagonists and to avoid sorting out highly effective strains, it is crucial to consider different experimental approaches since the efficacy might differ substantially depending on the incubation conditions. By using two distinct experimental set-ups, we identified promising biological control agents. SIGNIFICANCE AND IMPACT OF THE STUDY: FHB is one of the most devastating fungal cereal diseases worldwide. As the pathogen overwinters on crop residues, application of antagonists on residues of the previous crop during harvest could be a promising approach to efficiently control FHB in cereals as an essential part of an integrated disease management.

Chronic inflammation and protection from acute hepatitis in transgenic mice expressing TNF in endothelial cells.

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-gamma were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.

Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay.

We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.

Excision and disassembly of sperm tail microtubules during sea urchin fertilization: requirements for microtubule dynamics.

To determine the fate of the sperm tail during fertilization, the microtubules of the incorporated axoneme are measured using a monoclonal antibody against acetylated alpha-tubulin in zygotes from the sea urchin Strongylocentrotus purpuratus. This antibody recognizes axonemal microtubules, but does not recognize egg cytoplasmic tubulin or microtubules. The detachment of the axoneme from the male pronucleus occurs as early as 15 min post-insemination. Following excision, the axoneme is often found in close association with the female pronucleus during its migration to the male pronucleus. Fragmentation of the sperm tail, detected at 25 min, continues with only a few micrometers remaining at 85 min post-insemination. The fluorescence intensity of the axonemal fragments diminishes over time as compared to intact axonemes. At 100 min post-insemination, the sperm axoneme is no longer detected. Alternative imaging approaches using brief cold or elevated calcium extraction to disrupt the labile cytoplasmic, but not axonemal, microtubules, indicate that these observations are not due to changes in the post-translational modifications of alpha-tubulin. In the presence of nocodazole, a microtubule assembly inhibitor, a large portion of the tail remains visible at 100 min post-insemination; this suggests that microtubule dynamics are required for the disassembly of the sperm tail. Furthermore, the detachment of the axoneme from the male pronucleus requires the formation of the sperm aster. This suggests that the sperm aster microtubules both detach the axoneme from the male pronucleus, and also cause the translocation of the tail towards the female pronucleus after pronuclear union. In summary, the sperm tail is excised from the male pronucleus and the tail microtubules disassembled during the first cell cycle of sea urchin fertilization, and these events require new microtubule assembly within the zygote.