Water flows in the parasitic association Rhinanthus minor/Hordeum vulgare

Using the facultative root hemiparasite Rhinanthus minor and its host Hordeum vulgare several aspects of water relations have been measured in this parasitic association. Extraction of xylem sap by the parasite from the host's roots is facilitated by con siderably higher transpiration per leaf area in the parasite than in the host and by the fact that stomata of attached Rhinanthus were open all day and night despite extremely high ABA concentrations in the leaves. By comparison, another root hemiparasite, Melampyrum arvense, parasitizing various grasses in the field, showed normal diurnal stomatal behaviour. The abnormal behaviour of Rhinanthus stomata was not due to anatomical reasons as closure could be induced by applying high external ABA concentrations. Remarkable differences have been detected between the hydraulic conductance of barley seminal roots showing relatively low values and that of Rhinanthus seminal roots showing very high values. The latter could be related to the observed high ABA concentrations in these roots. Whole plant water uptake, transpirational losses, growth-dependent deposition, and the flows of water within the plants have been measured in singly growing Rhinanthus and Hordeum plants and in the parasitic association between the two. Water uptake, deposition and transpiration in Rhinanthus were dramatically increased after attachment to the barley host; most of the water used by the parasite was extracted as xylem sap from the host, thereby scavenging 20% of the total water taken up by the host's roots. This water uptake by the parasitized host, however, due to a parasite-induced reduction in the host's growth, was decreased by 22% as compared to non-parasitized barley. The overall changes in growth-related water deposition in the host and parasite pointed to decreased shoot growth and relatively favoured root growth in the host and to strongly favoured shoot growth in the parasite. These changes in the host became more severe, when more than one Rhinanthus was parasitizing one barley plant.     

Comparison and validation of novel pyrogen tests based on the human fever reaction

The Limulus amoebocyte lysate (LAL) test has replaced about 80% of the use of the rabbit pyrogen test. Ideally, human-based in vitro tests are needed, to replace the remaining use of the rabbit test and the use of the LAL test. The progress of an EU-funded project is described, in which a number of in vitro tests, based on the human fever reaction are passing through a prevalidation study on the way to evaluation in a formal validation study.     

Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase

Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1beta and TNF-alpha are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1beta/TNF-alpha production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1beta/TNF-alpha production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1beta/TNF-alpha upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.     

Profound differences in leukocyte-endothelial cell responses to lipopolysaccharide versus lipoteichoic acid

We have investigated the effects of LPS from Escherichia coli, lipoteichoic acid (LTA), and peptidoglycan (PepG) from Staphylococcus aureus, and live S. aureus on leukocyte-endothelial interactions in vivo using intravital microscopy to visualize muscle microvasculature. Systemic vs local administration of LPS induced very different responses. Local administration of LPS into muscle induced significant leukocyte rolling, adhesion, and emigration in postcapillary venules at the site of injection. LPS given systemically dramatically dropped circulating leukocyte counts and increased neutrophils in the lung. However, the drop in circulating leukocytes was not associated with leukocyte sequestration to the site of injection (peritoneum) nor to peripheral microvessels in muscles. Unlike LPS, various preparations of LTA had no systemic and very minor local effect on leukocyte-endothelial interactions, even at high doses and for prolonged duration. LPS, but not LTA, potently activated human endothelium to recruit leukocytes under flow conditions in vitro. Endothelial adhesion molecule expression was also increased extensively with LPS, but not LTA. Interestingly, systemic administration of live S. aureus induced leukocyte-endothelial cell responses similar to LPS. PepG was able to induce leukocyte-endothelial interactions in muscle and peritoneum, but had no effect systemically (no increase in neutrophils in lungs and no decrease in circulating neutrophil counts). These results demonstrate that: 1) LPS has potent, but divergent local and systemic effects on leukocyte-endothelial interactions; 2) S. aureus can induce a systemic response similar to LPS, but this response is unlikely to be due to LTA, but more likely to be mediated in part by PepG.     

Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to D-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2(-/-) mice was associated with normal levels of IL-1 beta, IL-6, and IFN-gamma, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1 beta, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2(-/-) mice displayed diminished IFN-gamma synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-gamma production required for activation of antibacterial effector mechanisms.     

Intron gain and loss in the evolution of the conserved eukaryotic recombination machinery

Intron conservation, intron gain or loss and putative intron sliding events were determined for a set of three genes (SPO11, MRE11 and DMC1) involved in basic aspects of recombination in eukaryotes. These are ancient genes and present in nearly all of the major kingdoms. MRE11 is of bacterial origin and can be found in all kingdoms. DMC1 is a specialized homolog of the bacterial RecA protein, whereas the SPO11 gene is of archaebacterial origin. Only unique homologs of SPO11 are found in animals and fungi whereas three distantly related SPO11 copies are present in plant genomes. A comparison of the respective intron positions and phases of all genes was performed, demonstrating that a quarter of the intron positions were perfectly conserved over more than 1000000000 years. Regarding the remaining three quarters of the introns we found insertions to be about three times more frequent than deletions. Aligning the introns of the three different SPO11 homologs of Arabidopsis thaliana we propose a conclusive model of their evolution. We postulate that at least one duplication event occurred shortly after the divergence of plants from animals and fungi and that a respective homolog has been retained in a protist group, the apicomplexa.     

Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid,auxin, cytokinin,ethylene and abscisic acid

The development of Agrobacterium tumefaciens-induced plant tumors primarily depends on the excessive production of auxin and cytokinin by enzymes encoded on T-DNA genes integrated into the plant genome. The aim of the present study was to investigate the involvement of additional phytohormone signals in the vascularization required for rapid tumor proliferation. In stem tumors of Ricinus communis L., free auxin and zeatin riboside concentrations increased within 2 weeks to 15-fold the concentrations in control stem tissue. Auxin and cytokinin immunolocalization revealed the highest concentrations within and around tumor vascular bundles with concentration gradients. The time-course of changes in free auxin concentration in roots was inversely correlated with that in the tumors. The high ethylene emission induced by increased auxin- and cytokinin correlated with a 36-fold accumulation of abscisic acid in tumors. Ethylene emitted from tumors and exogenously applied ethylene caused an increase in abscisic acid concentrations also in the host leaves, with a diminution in leaf water vapor conductance. Jasmonic acid concentration reached a maximum already within the first week of bacterial infection. A wound effect could be excluded. The results demonstrate the concerted interaction of a cascade of transiently induced, non-T-DNA-encoded phytohormones jasmonic acid, ethylene and abscisic acid with T-DNA-encoded auxin and zeatin riboside plus trans-zeatin, all of which are required for successful plant tumor vascularization and development together with inhibition of host plant growth.