A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

The diversity of mycorrhizal fungi in Japanese Cephalanthera species

The diversity of mycorrhizal fungi among four Japanese Cephalanthera (Orchidaceae) species was examined at a total of seven sites, based on sequence variation in nuclear ribosomal DNA. This is the first report to detect mycorrhizal fungi in C. subaphylla. Two patterns of mycorrhizal associations were confirmed from our results. C. falcata has lower fungal specificity, associating with Russulaceae, Sebacinales and Thelephoraceae. By contrast, the three Cephalanthera species C. erecta, C. subaphylla, and C. longifolia were associated mainly with Thelephoraceae fungi. There is no large difference found in mycobionts between green and albino individuals of C. falcata and no distinct preference of Japanese Cephalanthera species for a specific fungal group of Thelephoraceae.     

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn 10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10 -flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB . Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.     

CCL3 and viral chemokine-binding protein gg modulate pulmonary inflammation and virus replication during equine herpesvirus 1 infection

CCL3 is a proinflammatory chemokine that mediates many of the cellular changes occurring in pulmonary disease. Here, CCL3^−/− mice were used to investigate the role of this chemokine during respiratory herpesvirus infection. Compared to wild-type mice, CCL3^−/− mice infected with the alphaherpesvirus equine herpesvirus 1 (EHV-1) displayed reduced body weight loss but had higher pulmonary viral loads. Lungs from infected CCL3^−/− mice suffered a milder interstitial pneumonia, and fewer immune cells were recovered from the pulmonary airways after infection. We could also demonstrate that herpesvirus-encoded chemokine-binding glycoprotein G (gG) was capable of inhibiting the chemotactic functions of CCL3. This CCL3-mediated chemotaxis, however, was restored in the presence of gG-specific antibodies, which puts into question the advertised use of gG deletion mutants as marker vaccines. In summary, we concluded that CCL3 is a major player in controlling herpesvirus replication in the target organ, the lung, and does so by evoking a strong inflammatory response. The immunomodulatory activity of CCL3 is balanced by the expression of viral gG, whose chemokine-binding activity is mitigated in secondary infections by the production of anti-gG antibodies.     

Change in biomass of symbiotic ants throughout the ontogeny of a myrmecophyte, Macaranga beccariana (Euphorbiaceae)

Macaranga myrmecophytes (ant-plants) provide their partner symbiotic ants (plant-ants) with food bodies as their main food, and they are protected by the plant-ants from herbivores. The amount of resource allocated to food bodies determines the plant-ant colony size and consequently determines the intensity of ant defense (anti-herbivore defense by plant-ants). As constraints in resource allocation change as plants grow, the plant-ant colony size is hypothesized to change with the ontogenesis of Macaranga myrmecophyte. To determine the ontogenetic change in the relative size of the plant-ant colony, we measured the dry weights of the whole plant-ant colony and all of the aboveground parts of trees at various ontogenetic stages for a myrmecophytic species (Macaranga beccariana) in a Bornean lowland tropical rain forest. Ant biomass increased as plant biomass increased. However, the rate of increase gradually declined, and the ant biomass appeared to reach a ceiling once trees began to branch. The ant/plant biomass ratio consistently decreased as plant biomass increased, with the rate of decrease gradually accelerating. We infer that the ontogenetic reduction in ant/plant biomass ratio is caused by an ontogenetic change in resource allocation to food rewards for ants related to the physiological changes accompanying the beginning of branching.     

Maternal exercise training attenuates endotoxin-induced sepsis in mice offspring

Regular exercise during pregnancy can prevent offspring from several diseases, such as cardiovascular diseases, obesity, and type II diabetes during adulthood. However, little information is available about whether maternal exercises during pregnancy protect the offspring from infectious diseases, such as sepsis and multiple organ dysfunction syndrome (MODS). This study aimed to investigate whether maternal exercise training protects the offspring from endotoxin-induced septic shock in mice. Female C57BL/6 mice performed voluntary wheel exercises during pregnancy. All dams and offspring were fed normal chow with sedentary activity during lactation and after weaning. At 10-week-old, mice were intraperitoneally injected a lethal (30 mg/kg) or nonlethal (15 mg/kg) dose of lipopolysaccharide (LPS), following which the survival of mice that were administered a lethal dose was monitored for 60 h. Plasma, lung, and liver samples were collected 18 h after the injection to evaluate the cytokine concentration or mRNA expression from those administered a nonlethal dose. Although maternal exercise training could not prevent lethality during an LPS-induced septic shock, it significantly inhibited the LPS-induced loss of body weight in female offspring. Regular maternal exercise significantly inhibited the mRNA expression of the LPS-induced inflammatory cytokines, such as interleukin-1β (IL-1β) and interferon-γ (IFN-γ), in the plasma and liver. Thus, maternal exercise inhibited the LPS-induced inflammatory response in female offspring, suggesting that regular exercise during pregnancy could be a potential candidate of the onset of sepsis and MODS in offspring.