Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.     

Trade-offs in the vertical distribution of zooplankton: ideal free distribution with costs?

Zooplankton vertical migratory patterns are a classic example of optimal habitat choice. We hypothesize that zooplankton distribute themselves vertically in the water column according to an ideal free distribution (IFD) with costs such as to optimize their fitness. In lakes with a deep-water chlorophyll maximum, zooplankton are faced with a trade-off, either experiencing high food (high reproductive potential) but low temperature (slow development) in the hypolimnion or high temperature and low food in the epilimnion. Thus, in the absence of fish predation (e.g. at night) they should allocate the time spent in the different habitats according to fitness gain dependent on the temperature gradient and distribution of food. We tested this hypothesis with a Daphnia hyalina x galeata clone in large indoor columns (Plön Plankton Towers) and with a dynamic energy budget model. In the tower experiments, we simulated a deep-water algal maximum below the thermocline with epilimnetic/hypolimnetic temperature differences of 2, 5 and 10 degrees C. Experimental data supported the model. We found a significantly larger proportion of daphniids in the hypolimnion when the temperature difference was smaller. Our results are consistent with the concept of IFD with costs originally developed for stream fishes. This concept can be applied to predict the vertical distribution of zooplankton in habitats where fish predation is of minor importance.     

B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-alpha, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.     

Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the role of a putative composite transposon containing scpB and lmb

Beta-haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16 kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.     

Multi-step control of spindle pole body duplication by cyclin-dependent kinase

Organelles called centrosomes in metazoans or spindle pole bodies (SPBs) in yeast direct the assembly of a bipolar spindle that is essential for faithful segregation of chromosomes during mitosis. Abnormal accumulation of multiple centrosomes leads to genome instability, and has been observed in both tumour cells and cells with targeted mutations in tumour-suppressor genes. The defects that lead to centrosome amplification are not understood. We have recapitulated the multiple-centrosome phenotype in budding yeast by disrupting the activity of specific cyclin-dependent kinase (CDK) complexes. Our observations are reminiscent of mechanisms that govern DNA replication, and show that specific cyclin/CDK activities function both to promote SPB duplication and to prevent SPB reduplication.     

Analysis of right ventricular function in healthy mice and a murine model of heart failure by in vivo MRI

Because of its complex geometry, assessment of right ventricular (RV) function is more difficult than it is for the left ventricle (LV). Because gene-targeted mouse models of cardiomyopathy may involve remodeling of the right heart, the purpose of this study was to develop high-resolution functional magnetic resonance imaging (MRI) for in vivo quantification of RV volumes and global function in mice. Thirty-three mice of various age were studied under isoflurane anesthesia by electrocardiogram-triggered cine-MRI at 7 T. MRI revealed close correlations between RV and LV stroke volume and cardiac output (r = 0.97, P < 0.0001 each). Consistent with human physiology, murine RV end-diastolic and end-systolic volumes were significantly higher compared with LV volumes (P < 0.05 each). MRI in mice with LV heart failure due to myocardial infarction revealed significant structural and functional changes of the RV, indicating RV dysfunction. Hence, MRI allows for the quantification of RV volumes and global systolic function with high accuracy and bears the potential to evaluate mechanisms of RV remodeling in mouse models of heart failure.     

Identification of tissue-specific microRNAs from mouse

MicroRNAs (miRNAs) are a new class of noncoding RNAs, which are encoded as short inverted repeats in the genomes of invertebrates and vertebrates. It is believed that miRNAs are modulators of target mRNA translation and stability, although most target mRNAs remain to be identified. Here we describe the identification of 34 novel miRNAs by tissue-specific cloning of approximately 21-nucleotide RNAs from mouse. Almost all identified miRNAs are conserved in the human genome and are also frequently found in nonmammalian vertebrate genomes, such as pufferfish. In heart, liver, or brain, it is found that a single, tissue-specifically expressed miRNA dominates the population of expressed miRNAs and suggests a role for these miRNAs in tissue specification or cell lineage decisions. Finally, a miRNA was identified that appears to be the fruitfly and mammalian ortholog of C. elegans lin-4 stRNA.