Lactobacillus fermentum BCS87 expresses mucus‐ and mucin‐binding proteins on the cell surface

Aims: To identify and characterize adhesion‐associated proteins in the potential probiotic Lactobacillus fermentum BCS87. Methods and Results: Protein suspensions obtained from the treatment of Lact. fermentum BCS87 with 1 mol 1^?1 LiCl were analysed by Western blotting using HRP‐labelled porcine mucus and mucin. Two adhesion‐associated proteins with relative molecular weight of 29 and 32 kDa were identified. The N‐terminal and internal peptides of the 32 kDa protein (32‐Mmubp) were sequenced, and the corresponding gene (32‐mmub) was found by inverse polymerase chain reaction. The complete nucleotide sequence of 32‐mmub revealed an open reading frame of 903 bp encoding a primary protein of 300 amino acids and a mature protein of 272 residues. A basic local alignment search showed 47–99% identity to solute‐binding components of ATP binding cassette transporter proteins in Lactobacillus, Streptococcus and Clostridium. An OpuAC‐conserved domain was identified and phylogenetic relationship analysis confirmed that 32‐Mmubp belongs to the OpuAC family. Conclusions: Adhesion of Lact. fermentum BCS87 appeared to be mediated by two surface‐associated proteins. 32‐Mmubp is a component of ABC transporter system that also functions as an adhesin. Significance and Impact of the Study: Characterization of 32‐Mmubp and 32‐mmub will contribute to understanding the host–bacteria interactions of Lact. fermentum with the intestinal tract of pigs.     

Skin structure in six dragonet species (Gobiesociformes; Callionymidae): interspecific differences in glandular cell types and mucus secretion

A comparative histological and histochemical study on the skin structure of six dragonet species, Synchiropus splendidus , Synchiropus picturatus, Synchiropus ocellatus , Repomucenus richardsonii , Callionymus decoratus and Callionymus risso showed that the epidermis of all species was rich in mucous cells (globlet cells), varying in distribution and elaboration among species. A second, distinctive, cell type, the sacciform cell, was also found in S. splendidus and S. picturatus . The secretions of these two cell types probably serve several functions, including those related to predation and the close contact that these species have with the substratum. The differences in type, elaboration and distribution of the cells suggested a pattern among the six callionymid species, in relation to habitat and body colouration differences.     

Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na(+) absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na(+), Mg(2+), P, S and Cl(-)) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR(inh)-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.     

Modeling the airway epithelium in allergic asthma: Interleukin-13-induced effects in differentiated murine tracheal epithelial cells

Summary Mucous cells of the airway epithelium play a crucial role in the pathogenesis of human inflammatory airway diseases. Therefore, it is of importance to complement in vivo studies that use murine models of allergic asthma with in vitro mechanistic studies that use murine airway epithelial cells, including mucus-containing cells. In this study, we report the development and characterization of an in vitro culture system for primary murine tracheal epithelial (MTE) cells comprising ciliated cells and a substantial number of mucous cells. The increase in mucous cell number over that observed in the native murine airway, or in previously described murine cultures, creates a culture intermediate between the in vivo murine airway epithelium and in vitro cultures of human airway epithelial cells. To establish the usefulness of this culture system for the study of epithelial effects during inflammatory airway diseases, the cells were exposed to interleukin (IL)-13, a central inflammatory mediator in allergic asthma. The IL-13 induced two characteristic epithelial effects, proliferation and modulation of MUC5AC gene expression. There was a concentration dependence of these events, wherein high concentrations of IL-13 (10 ng/ml) induced proliferation, whereas lower concentrations (1 ng/ml) increased MUC5AC mRNA (where mRNA is messenger RNA). Interestingly, these effects occurred in an inverse manner, with the high concentration of IL-13 also provoking a significant decrease in MUC5AC gene expression. Thus, MTE cells cultured in this manner may provide an important link between experimental findings from animal models of allergic asthma and their application to human disease.     

Influence of TNFalpha on the sialylation of mucins produced by a transformed cell line MM-39 derived from human tracheal gland cells

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNF. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNF; a 52% increase of 2,3-sialyltransferase activity was also observed in TNF-stimulated MM-39 cells. After metabolic radio-labelling with [³H]glucosamine and [³H]fucose, the mucins released inthe culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39–1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNF was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the 2,3-sialyltransferase activity by TNF argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNF. In conclusion, the influence of TNF on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.     

Mucus blocks probiotics but increases penetration of motile pathogens and induces TNF-α and IL-8 secretion

The mucosal barrier in combination with innate immune system are the first line of defense against luminal bacteria at the intestinal mucosa. Dysfunction of the mucus layer and bacterial infiltration are linked to tissue inflammation and disease. To study host–bacterial interactions at the mucosal interface, we created an experimental model that contains luminal space, a mucus layer, an epithelial layer, and suspended immune cells. Reconstituted porcine small intestinal mucus formed an 880 ± 230 µm thick gel layer and had a porous structure. In the presence of mucus, sevenfold less probiotic and nonmotile VSL#3 bacteria transmigrated across the epithelial barrier compared to no mucus. The higher bacterial transmigration caused immune cell differentiation and increased the concentration of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α; p < .01). Surprisingly, the mucus layer increased transmigration of pathogenic Salmonella and increased secretion of TNF-α and IL-8 (p < .05). Nonmotile, flagella knockout Salmonella had lower transmigration and caused lower IL-8 and TNF-α secretion (p < .05). These results demonstrate that motility enables pathogenic bacteria to cross the mucus and epithelial layers, which could lead to infection. Using an in vitro coculture platform to understand the interactions of bacteria with the intestinal mucosa has the potential to improve the treatment of intestinal diseases.     

The effects of proteins secreted by fibroblasts from patients with cystic fibrosis on hamster tracheal explants

Summary Hamster tracheal explants have been used to assay for mucosecretory activity in media taken from cultures of fibroblasts isolated from patients with cystic fibrosis (CF). Cystic fibrosis and normal sera were first used to establish optimal conditions for mucus release in the hamster tracheal ring assay. Unless protein levels were maintained at 5% serum concentration or greater there was loss of cilia, nonspecific mucus accumulation, and extensive epithelial damage to the luminal surface. Likewise, it was shown that exposure of the explants to unconcentrated conditioned media from CF (GM 770, 768, 1348, 142) or normal (GM 3349, 38) cultured fibroblasts for 1, 6, or 12 h resulted in the same type of damage and this was due to low protein levels. When the protein concentration of the conditioned media was increased with fetal bovine serum, the morphological integrity of the explants was maintained, demonstrating that there was no apparent difference between CF and normal fibroblast-secreted proteins in ability to induce mucus release. The ciliary inhibitory capacity of CF serum-derived or fibroblast-derived factor had been reported to require IgG for activity. However, addition of IgG to high molecular weight (VoP10) or low molecular weight (VeP10) secreted proteins had no apparent effect on stimulating secretion. In conclusion, it is possible that CF fibroblasts do not secrete a protein that has the mucostimulatory effect and thus these cells may not be suitable for studying the CF-related activity.     

The potential for population growth of Ascomorpha ecaudis

The intrinsic rate of natural increase (r_m) of the rotifer Ascomorpha ecaudis was found to be a hyperbolic function of food concentration. The threshold food concentration and r_max/2-food concentration determined for this species were significantly lower than values predicted from allometric models. These growth characteristics may be related to the mucus house in which Ascomorpha lives and/or the symbiotic algae living in its body tissues. The maximum rate of population growth recorded (0.71 d^–1) was similar to that of other soft-bodied rotifers of similar body mass. These population growth characteristics and the resistance of this species to invertebrate predation should allow it to become a dominant member of freshwater zooplankton communities. However, field observations suggest that it is not. Reasons for this are suggested.     

Biochemical changes in cervical mucus-factor infertility

Cervical-factor infertility has generally been attributed to the presence of antispermatozoal antibodies in the secretions of the uterine cervix, despite the fact that the incidence of sperm-specific antibodies in these women is generally low. We report here a modification in the structure of the cervical mucus of patients with a diagnosed cervical factor. Cervical mucus from patients with a cervical factor of nonimmunological origin, collected during the periovulatory phase of the menstrual cycle, had (1) a significant decrease in the content of glycosidically bound sialic acid and (2) an increased ability to act as an acceptor for sialic acid from cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-sialic acid) when incubated with an exogenous sialyltransferase; in comparison to mucus from normal fertile women. Both siaiyltransferase and fucosyltransferase activities were detected in cervical mucus, but there was no difference between fertile normal and cervical-factor patients using the assays described. These results reinforce a possible role for sialic acid residues and their associated glycosyltransferases in the regulation of spermatozoal–cervical mucus interaction.     

Studies on the Metabolism of Gallic Acid by Microorganisms

The intermediary metabolism of gallic acid by Aspergillus niger under the influence of some added inhibitors has been studied. The decomposition of gallic acid by lyophilized cells under fluoroacetate inhibition allowed cis-aconitic acid, α-ketoglutaric acid and citric acid to accumulate. A mechanism of gallic acid decomposition via cis-aconitic acid has been inferred.