Enhancement of death-receptor induced caspase-8-activation in the death-inducing signalling complex by uncoupling of oxidative phosphorylation

Signalling through the death receptor CD95 induces apoptosis by formation of a signalling complex at the cell membrane and subsequent caspase-8 and caspase-3-activation. Treatment of Jurkat T cells with protonophores across the mitochondrial membrane such as 2,4-dinitrophenol (DNP) enhances the death-inducing capacity of CD95. In this study, we show that this enhancement is due to the specific acceleration of caspase-8-processing and activation at the CD95-receptor. DNP-treatment did not affect NF-kappaB-induction by CD95. Immunoprecipitation experiments showed that the amounts of the adapter FADD/MORT1 and pro-caspase-8 at the CD95-receptor were not altered by DNP. Subcellular fractionation studies revealed that the amount of mature caspase-8 but not pro-caspase at the membrane was increased following CD95-stimulation in the presence of DNP. As a consequence of caspase-activation, c-FLIP-levels in the cytosol decreased. In Jurkat cells overexpressing c-FLIPS, DNP was still able to enhance caspase-activation. The enhancing capacity of DNP was seen in some cell lines (Jurkat, CEM and HeLa) but not in SKW6 cells and was also found in mitogen-stimulated human T cells. Furthermore, the enhancement extended to TRAIL-induced caspase-activation. Thus, a mechanism exists by which caspase-8-activation can be accelerated at death receptors and this mechanism can be triggered by targeting mitochondrial oxidative phosphorylation.     

Sleep and memory consolidation: The role of electrophysiological neuroimaging

Summary Memory consolidation involves a complex series of molecular, cellular and network-level processes that take place on time scales from millisecond to months. Evidence from a wide range of experimental observations supports the hypothesis that parts of these processes occur during sleep when the brain is not engaged in processing and encoding incoming information. Indeed, sleep seems to be favorable for brain plasticity. Experience-dependent cortical plasticity observed during sleep has been hypothesized to be part of the global process of memory consolidation. Thus, studying task-dependent, regionally specific reactivation of neuronal assemblies during posttraining sleep may make important contributions to elucidating the role of sleep in memory trace processing. A new methodology – low-resolution brain electromagnetic tomography (LORETA) – offers the possibility of localizing electrical activity produced by cortical neuronal generators under normal (undisturbed) sleeping conditions. The high time resolution of brain electrical data can be exploited to produce neuroimages for specific EEG spectral frequency bands (e.g. delta, theta, or spindle bands). This makes it possible to investigate, dependent on the type of memory, when – in which sleep stages (S2 sleep, SWS, REM sleep) – and where – in which cortical brain regions (primary sensory cortex, higher association cortex) – experience-dependent reactivation occurs.     

Light controlled solubility change of polymers: Copolymers of N,N-dimethylacrylamide and 4-phenylazophenyl acrylate

Copolymers of N,N-dimethylacrylamide (DMA) and 4-phenylazophenyl acrylate (PAPA) have been prepared by free-radical copolymerization in dioxane (DMAP-x). The molecular weights of the copolymers show a strong decrease with increasing azobenzene content due to the retardation effect of the azobenzene group. An analogous decrease in molecular weight was found with a number of polymers of DMA which were polymerized in the presence of the non-polymerizable model 4-phenylazophenyl propionate (PAPP). We were able to induce a lower critical solution temperature (LCST) above a certain content of azobenzene moieties in the copolymers (DMAP-x) in contrast to the good water solubility of poly(DMA). Due to the reversible photoisomerization and the concomitant change in the dipole moment of the azobenzene moieties, the LCST depends not only on the content of azobenzene but also on the degree of isomerization. A difference in the LCST up to 20°C was found for dark adapted polymer solutions (0% Z-isomer) and polymer solutions in the photostationary state (ca. 85% Z-isomer). Within this temperature range the polymer can be precipitated by irradiation with light.     

Characterization of a population of small macrophages in induced sputum of patients with chronic obstructive pulmonary disease and healthy volunteers

The inflammatory process in chronic obstructive pulmonary disease (COPD) is active mainly in the airways, but little is known about the properties of the inflammatory cells in this compartment. We have studied leucocytes in induced sputum of COPD patients compared to controls in order to uncover what types of macrophages might be involved in the disease. Sputum induction was performed by inhalation of nebulized sodium chloride solution. Leucocytes were isolated and stained with specific monoclonal antibodies for analysis in flow cytometry. Flow cytometry analysis revealed that a major portion of CD14+ macrophages in COPD has lower forward scatter, i.e. they are small macrophages. While in control donors these small macrophages accounted for 6.9% of all macrophages, the percentage of these cells in COPD was 45.7%. CD14 and HLA-DR expression was high on these small sputum macrophages while the large sputum macrophages expressed only low levels of these surface molecules, both in control donors and COPD patients. Small sputum macrophages of both control donors and COPD patients showed higher levels of constitutive tumour necrosis factor (TNF) compared to the large macrophages. TNF was inducible by lipopolysaccharide (LPS) preferentially in the small sputum macrophages in the control donors but there was no further induction in COPD patients. These data show that the small sputum macrophages are a major macrophage population in COPD and that these cells exhibit features of highly active inflammatory cells and may therefore be instrumental in airway inflammation in COPD.     

Thyroid hormone promotes neurogenesis in the Xenopus spinal cord.

Three phases of neurogenesis can be recognized during Xenopus spinal cord development. An early peak during gastrulation/neurulation is followed by a phase of low level neurogenesis throughout the remaining embryonic stages and a later peak at early larval stages. We show here that several genes known to be essential for early neurogenesis (X-NGNR-1, XNeuroD, XMyT1, X-Delta-1) are also expressed during later phases of neurogenesis in the spinal cord, suggesting that they are involved in regulating spinal neurogenesis at later stages. However, additional neuronal determination genes may be important during larval stages, because X-NGNR-1 shows only scant expression in the spinal cord during larval stages. Thyroid hormone treatment of early larvae promotes neurogenesis in the spinal cord, where thyroid hormone receptor xTRalpha is expressed from early larval stages onward and results in precocious up-regulation of XNeuroD, XMyT1, and N-Tubulin expression. Similarly, thyroid hormone treatments of Xenopus embryos, which were coinjected with xTRalpha and the retinoid X receptor xRXRalpha, repeatedly resulted in increased numbers of neurons, whereas unliganded receptors repressed neurogenesis. Our findings show that thyroid hormones are sufficient to up-regulate neurogenesis in the Xenopus spinal cord.     

Disseminated infection with Nattrassia mangiferae in an immunosuppressed patient

Disseminated infection with the coelomycetous fungus Nattrassia mangiferae is a very rare disease affecting only the immunocompromised host. We report the first case of a disseminated infection with spondylodiscitis and granular skin lesions due to N. mangiferae in a renal transplant patient.     

Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55 degrees C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.     

Role of the alpAB proteins and lipopolysaccharide in adhesion of Helicobacter pylori to human gastric tissue

The attachment of the bacterial pathogen Helicobacter pylori (H. pylori) to gastric epithelial cells is commonly believed to be required for an efficient and persistent colonization of the human stomach as well as for host cell trans-membrane signaling. In the past, several putative adhesins were postulated, including the outer membrane proteins AlpAB and the bacterial lipopolysaccharide (LPS) presenting oligomeric Lewis x (Le(x)) sugar components. We investigated the AlpAB-mediated and the Le(x)-dependent binding by knockout mutagenesis in one distinct strain, H. pylori P1. We show here that the mutagenesis of either alpA and/or alpB dramatically reduced the adherence of H. pylori P1 to a given gastric biopsy section. None of these mutations influenced the surface exposure of the Le(x) antigen, suggesting that the assembly and/or presentation of LPS is independent of the AlpAB outer membrane proteins. However, a truncation of the LPS O-side chain by a galE mutation abolished the presentation of the Le(x) epitope. This Le(x)-negative strain did not show any significant reduction in its binding capacity to the gastric tissue, when compared with the corresponding wild-type strain. From these data we conclude that the AlpAB-specific adherence is independent of the composition of the LPS and that the oligomeric Le(x) structure does not confer binding to the gastric biopsy material used in this study. As the adhesion properties of our H. pylori strain P1 vary in dependence on the respective biopsy donor it is assumed that the surface-exposed Le(x) epitope recognizes a different host cell receptor than AlpAB, which was probably not present in the tissue sections used in this study.