Reconsidering environmental effects assessment of chemicals: Proposal for a dynamic testing strategy

Certain substances may be hazardous to ecosystems. To be able to preserve the structures and functions of ecosystems, knowledge is required to qualify and quantify such hazards. To this end, biotests are indispensable tools. For the development and/or choice of biotests, special attention has to be drawn to conflicts between scientific demands and practical constraints. From a purely scientific point of view, experiments should be designed to maximise the ecological relevance of the obtained results. However, this often collides with the limited resources (budget, time, manpower) available. Furthermore, societal issues (e.g. animal welfare) have to be taken into account. Thus, it is necessary to develop a scientifically sound testing approach that avoids unnecessary animal testing, keeps the costs low, and can be performed within a short timeframe. The different perspectives of ecology, environmental toxicology, and environmental chemistry should be integrated into a balanced ecotoxicological approach. Accordingly, we propose a dynamic testing strategy, which is adapted to the substance (or substance group) in question and its mode(s) of action.     

Characterization of nonstructural protein membrane anchor deletion mutants expressed in the context of the hepatitis C virus polyprotein

Protein-protein interactions involved in formation of the membrane-associated hepatitis C virus (HCV) replication complex are poorly understood. Here, we investigated nonstructural proteins with deletions in their membrane anchor domains when expressed in the context of the entire HCV polyprotein. Interactions among cytosolic domains of HCV nonstructural proteins were found not to be sufficiently strong to rescue such mutants to the membrane. Thus, the membrane anchor domains of nonstructural proteins are essential for incorporation of these proteins into the HCV replication complex while interactions among the cytosolic domains appear to be relatively weak. This feature may provide the nonstructural proteins with a certain flexibility to perform their multiple functions during HCV replication.     

Experimental Helicobacter pylori infection induces antral-predominant, chronic active gastritis in hispid cotton rats (Sigmodon hispidus)

BACKGROUND: The hispid cotton rat has proven to be an excellent animal model for a variety of human infectious disease agents. This study was performed to evaluate the use of the cotton rat as a model of Helicobacter pylori infection. MATERIALS AND METHODS: Thirty-eight inbred cotton rats were orogastrically inoculated with a human strain of H. pylori. Twenty-eight control cotton rats were dosed with vehicle only. Animals were sacrificed at 2, 4, 12, 26, or 38 weeks after inoculation for bacterial and histologic and immunologic examinations. RESULTS: Helicobacter pylori was cultured from the glandular stomach of 89% of the infected cotton rats. The level of colonization was consistently high (approximately 10(4-6) colony-forming units/g tissue). Histologically, the spiral bacteria were demonstrated on the epithelial surface and in the foveolae of the gastric mucosa with highest numbers in the antrum. H. pylori infection was associated with antral-predominant, chronic active gastritis which progressively increased in severity over time. By week 26 of infection, moderate antral gastritis had developed with frequent involvement of the submucosa and formation of lymphocytic aggregates. Splenic T cells from infected cotton rats expressed mRNAs for interferon-gamma, interleukin-4, interleukin-6, and interleukin-10 following in vitro stimulation with H. pylori. Serum levels of H. pylori-specific immunoglobulin G were significantly elevated after 12 weeks of infection. CONCLUSIONS: The H. pylori-infected cotton rat represents a novel animal model that should prove useful for studies of H. pylori-induced chronic active gastritis and factors affecting gastric colonization by this pathogen.     

Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current

We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin-sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor-mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.     

G protein-coupled receptor 83 overexpression in naive CD4+CD25- T cells leads to the induction of Foxp3+ regulatory T cells in vivo

Foxp3 functions as a lineage specification factor for the development of naturally occurring thymus-derived CD4+CD25+ regulatory T (Treg) cells. Recent evidence suggests that naive Foxp3-CD4+CD25- T cells can be converted in the periphery into Foxp3+ Treg cells. In this study, we have identified the G protein-coupled receptor (GPR)83 to be selectively up-regulated by CD4+CD25+ Treg cells of both murine and human origin in contrast to naive CD4+CD25- or recently activated T cells. Furthermore, GPR83 was induced upon overexpression of Foxp3 in naive CD4+CD25- T cells. Transduction of naive CD4+CD25- T cells with GPR83-encoding retroviruses did not confer in vitro suppressive activity. Nevertheless, GPR83-transduced T cells were able to inhibit the effector phase of a severe contact hypersensitivity reaction of the skin, indicating that GPR83 itself or GPR83-mediated signals conferred suppressive activity to conventional CD4+ T cells in vivo. Most strikingly, this in vivo acquisition of suppressive activity was associated with the induction of Foxp3 expression in GPR83-transduced CD4+ T cells under inflammatory conditions. Our results suggest that GPR83 might be critically involved in the peripheral generation of Foxp3+ Treg cells in vivo.     

Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.     

Structure-functional analysis of the Dictyoglomus cell envelope

Several closely related strains of the thermophilic bacterium Dictyoglomus have been isolated from various hot springs on the Philippine archipelago. These strains as well as Dictyoglomus thermophilum H-6-12 were analyzed in view of the structure-functional relationships of the cell envelopes. All envelopes of Dictyoglomus strains show several peculiar features that are apparently either unique for the genus or common for other phylogenetically related Thermotogales. The filamentous cells develop pili at the cell poles, guided by large columnar protein assemblies that traverse the periplasm. Filamentous protein complexes span the periplasmic space at the longitudinal sides of the cell. By the end of the exponential growth phase, Dictyoglomus strains form multicellular aggregates ("rotund bodies") inside a compartment surrounded by a single, continuous outer envelope. The formation of these rotund bodies which are also found in some other deeply branching thermophilic phyla (Thermotoga, Thermus) was studied in detail. The transition between unicellular and multicellular lifestyle can be explained by the partial detachment of a protoplast from the outer envelope during cell division. When the outer envelope is partially detached from the protoplast, mechanical forces generated by protoplast elongation may drive cell rearrangement of daughter cells inside the compartment. During the following rounds of cell division, the overall shape of the compartment changes from spindle-like to globular geometry. Analysis of subcellular fractions of Dictyoglomus cells shows that glucan hydrolases are associated with the compartment. This feature is discussed in view of the multicellular life style of Dictyoglomus.     

Usefulness of the Serum Galactomannan Assay for Early Response Assessment and Treatment Stratifications of Invasive Aspergillosis

The galactomannan (GM) Enzyme Immunoassay (EIA) is an upcoming tool not only for diagnosis but also monitoring of invasive Aspergillosis (IA). Various studies were performed over the last years to apply such a promising instrument correctly. New findings show the potential of this test to segregate affected patients into treatment responders and non-responders at a time point as early as 7–14 days after initiation of antifungal therapy. Current data suggest that serial GM testing in patients receiving antifungal therapy for IA is essential as GMI kinetics may offer the clinician a substantial support in decision making concerning early therapeutic stratifications. The correct interpretation of GM EIA results with respect to the individual context of the patient is, however, absolutely necessary. The following review shall give an overview about the GM-EIA as a tool for IA monitoring and therapy stratification.     

Fbxw7 controls angiogenesis by regulating endothelial notch activity

Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific Fbxw7(iECKO) mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of fbxw7 morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs). Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature.