Study of the parietal microflora in the rat intestine

The article deals with the adaptation of the existing method of the isolation of microorganisms from parietal mucin of the intestine of experimental animals (rats) with a view to ensure the methodological uniformity of investigations. The effectiveness of the method of sparing disintegration of parietal mucin taken from the mucous membrane of the gastrointestinal tract, specially developed for human biomaterial, and its adequacy for microbiological investigations of the digestive organs of rats have been confirmed. A certain similarity between the microbiocenosis of the parietal mucin in the intestine of humans and rats has been established.     

Comparative study of parietal and lumen microflor in the large intestine in experiments on mice

The methodological aspects of the study of the microflora of the parietal mucous layer (parietal mucin) of the large intestine is discussed; the study is difficult because the mucin layer is thin closely associated with epithelium and the impossibility of the mechanical separation of this layer from the epithelium without damaging the latter is possible. The method of homogenization fails to determine of the composition of parietal mucin proper. Such possibility becomes real after the dissolution of mucin and obtaining the suspension of microbial cells. As experimentally shown in vitro, urea solution, reducing disulfide bonds, effectively depolymerized mucin and exhibited no antibacterial and cytolytic activity. With the use of urea treatment, microflora of parietal mucin of the large intestine was studied on 44 non-inbred mice. This newly developed method was shown to have higher resolution in comparison with the traditional one (homogenization). Some qualitative and quantitative characteristics of the microflora of parietal mucin were established. In the same group of mice the study of fecal microflora was made and compared with microflora of parietal mucin.     

Microbiocenosis of parietal mucin in gastrointestinal tract of rats with induced disbiosis

Comparative study of qualitative and quantitative characteristics of parietal mucin microbiocenosis in rats with alimentary induced disbiosis was performed. It has been shown that disbiosis was associated with changes in parietal microflora which were observed mainly in colon mucosa. Disbiosis in parietal area, in contrast to central area manifested by changes in quantitative characteristics of microbiocenosis. Results of the study allowed to suppose that parietal mucosal microbiocenosis was more stable than gut one and that this stability was determined by endogenous homeostatic factors of the host.     

Microbiocenosis of parietal mucin in the gastrointestinal tract of rats

The qualitative and quantitative composition of the microbial community in parietal mucin at different areas of the gastrointestinal tract (GIT) of rats was revealed. The pronounced variability in the quantitative and qualitative characteristics of microbiocenosis in parietal mucin of rats at different sections was revealed. The differences were most pronounced in the passage from upper to lower GIT sections, the large intestine found to be the richest biocenosis. The microbial composition of rat feces was faintly associated with the GIT parietal microbiocenosis. The individual areas of GIT mucosa were unique of their microbial characteristics and organization. This makes it possible to regard them as relatively independent biotopes and indicates that it is impossible to evaluate the microbial community by one of the colonic mucosal sifes.     

Microecology and humoral immunity characteristics of the vagina in women with nonspecific inflammatory diseases of the genitals

The complex cytological, microbiological and immunological examination of 90 female patients with nonspecific inflammatory diseases of the genital tracts and 30 clinically healthy women (the control group) was carried out. The examination revealed significant microbiocenosis changes in the lumen and the parietal area of the vagina, depending on the severity of the infectious process: a decrease in the level of lactobacilli, an increase in the content of opportunistic facultative anaerobic microorganism in the lumen and obligate anaerobic bacteria in the parietal area. Pronounced correlation between the content of opportunistic microorganisms and the levels of IgM and IgA, as well as secretory IgA and free secretory component in vaginal secretions in patients in patients with non-specific inflammatory diseases of the genital tracts has been revealed.     

Influence of Bacillus cereus on microbiocenosis of gastrointestinal tract in rats

The modifying effect of Bacillus cereus on intestinal microbiocenosis was investigated in eubiotic and disbiotic female rats. Qualitative and quantitative characteristics of gut and mucosal microflora from different parts of rats' intestine were studied before and after intragastral application of B. cereus suspension. The single application of B. cereus suspension resulted in appearance of this bacterium in feces and in parietal mucin from all parts of the intestine. In eubiotic rats compared with disbiotic, B. cereus adhere to parietal mucin much more efficiently and supplanted indigenous microflora. During disbiosis B. cereus sometimes had stimulating effect on the intestinal microbiocenosis. Gut microbiocenosis appeared to be more resistant to B. cereus invasion than mucosal. This fact was considered to be the evidence of higher sensitivity of mucosal microbiocenosis to short-term influence of exogenous microbial factor.     

Heterogeneity in gastrointestinal mucins

Pig digestive tract mucins have often been used as model mucins for studying mucin structure, function and metabolism. In the present study pig gastric mucin and pig colonic mucin in the subunit form have been characterised and compared. Following Sepharose 4B or 2B-CL gel chromatography, the mucin eluant fractions were assayed colorimetrically by both the periodic acid-Schiff and the Alcian blue binding assays. Subunit colonic mucin eluted as a single unimodel peak that was easily detected by both assays. In contrast, subunit gastric mucin gave a peak primarily detected by periodic acid-Schiff that was overlapped by, but partially separated from, another peak primarily detected by Alcian blue. Subunit gastric mucin was separated into two periodic acid-Schiff staining spots when electrophoresed on cellulose acetate. Cetylpyridinium chloride (CPC) was able to precipitate only about half the subunit gastric mucin. The CPC-precipitable subunit gastric mucin corresponded to the faster running spot on electrophoresis, and the subunit gastric mucin in the CPC supernatant (which may have more than one subunit mucin type) to the slower spot(s). The former had a higher sulphate content and stained with Alcian blue. The latter had a lower sulphate content and showed very little Alcian blue reactivity. These results indicate that subunit pig gastric mucin is heterogeneous with respect to both size and charge. The differences between the types may be important in biological and physiochemical behaviour of gastric mucin. It seems likely that different laboratories may have worked on one or other of the pig gastric mucin types or a mixture, depending on the preparation method.     

Polyethylene glycol-enhanced nephelometric quantification of bovine submaxillary mucin with Helix pomatia lectin

A new principle for quantification of intact mucin has been developed. The method is reproducible, simple, and sensitive at the submicrogram level and can be performed within an hour. The procedure involves incubation of mucin (bovine submaxillary mucin) and polyethylene glycol with the lectin from the snail Helix pomatia and the complexes formed are quantitated by nephelometry. Polyethylene glycol was found to enhance the affinitin reaction in a dose-dependent fashion. Increasing concentrations of acetate or citrate led to decreasing light scattering of the polyethylene glycol--bovine submaxillary mucin--H. pomatia lectin reaction mixture. Results from displacement studies with N-acetyl-galactosamine suggest that H. pomatia lectin may contain an allosteric type of regulatory site. The quantitative results obtained with bovine submaxillary mucin using the new method correlated well with those obtained with an established method for neuraminic acid determination after neuraminidase digestion (R = 0.969, P less than 0.001; n = 24).     

Bioinformatic identification of polymerizing and transmembrane mucins in the puffer fish Fugu rubripes

Mucins are large glycoproteins characterized by mucin domains that show little sequence conservation and are rich in the amino acids Ser, Thr, and Pro. To effectively predict mucins from genomic and protein sequences obtained from genome projects, we developed a strategy based on the amino acid compositional bias characteristic of the mucin domains. This strategy is combined with an analysis of other features commonly found in mucins. Our method has now been used to predict mucins in the puffer fish Fugu rubripes that were previously not identified or annotated. At least three gel-forming mucins were found with the same general domain structure as the human MUC2 mucin. In addition one transmembrane mucin was identified with SEA and EGF domains as found in the mammalian transmembrane mucins. These results suggest that the number of gel-forming mucins has been conserved during evolution of the vertebrates, whereas the family of transmembrane mucins has been markedly expanded in the higher vertebrates.     

TNF-alpha in the regulation of MUC5AC secretion: some aspects of cytokine-induced mucin hypersecretion on the in vitro model

TNF-alpha has been implicated in the aetiology of otitis media with effusion (OME), where goblet cells proliferate in a modified respiratory epithelium, leading to the accumulation of a mucin-rich effusion in the middle-ear cleft. The MUC5AC mucin gene product has been identified as a component of these effusions. Here we have used the HT29-MTX goblet cell line, which secretes MUC5AC mucin, as a model to study the effect of TNF-alpha on goblet cells. MUC5AC mucin was identified and quantitated with a monoclonal antibody NCL-HGM-45M1. TNF-alpha stimulates MUC5AC mucin secretion in a dose-dependent manner, with 20 ng/ml producing maximal stimulation. Both pre-confluent and confluent cells showed peak stimulation after 7 h, however the pre-confluent cells showed twice the level of mucin hypersecretion. These results suggest that TNF-alpha stimulation of mucin secretion could play an important role in the early acute phase of the development of OME. This hypersecretion of mucin could then lead to the failure of the mucociliary clearance system, resulting in the accumulation of a mucin-rich effusion in the middle ear and the movement to a more chronic phase of the disease.     

TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression

Excessive airway mucin production contributes to airway obstruction in lung diseases such as asthma and chronic obstructive pulmonary disease. Respiratory infections, such as atypical bacterium Mycoplasma pneumoniae (Mp), have been proposed to worsen asthma and chronic obstructive pulmonary disease in part through increasing mucin. However, the molecular mechanisms involved in infection-induced airway mucin overexpression remain to be determined. TLRs have been recently shown to be a critical component in host innate immune response to infections. TLR2 signaling has been proposed to be involved in inflammatory cell activation by mycoplasma-derived lipoproteins. In this study, we show that TLR2 signaling is critical in Mp-induced airway mucin expression in mice and human lung epithelial cells. Respiratory Mp infection in BALB/c mice activated TLR2 signaling and increased airway mucin. A TLR2-neutralizing Ab significantly reduced mucin expression in Mp-infected BALB/c mice. Furthermore, Mp-induced airway mucin was abolished in TLR2 gene-deficient C57BL/6 mice. Additionally, Mp was shown to increase human lung A549 epithelial cell mucin expression, which was inhibited by the overexpression of a human TLR2 dominant-negative mutant. These results clearly demonstrate that respiratory Mp infection increases airway mucin expression, which is dependent on the activation of TLR2 signaling.     

A simple enzymatic method for the analysis of macromolecular fucose in biological materials

A sensitive enzymatic method employing l-fucose dehydrogenase has been used for the measurement of the amounts of fucose in 100–500 μg of plasma membrane protein and 10–100 μg of porcine submaxillary mucin. The assay showed linearity between 0 and 20 nmol of α-l-fucose when measuring NADH fluorescence. The fucose values obtained for the plasma membrane and submaxillary mucin correspond well with those obtained by gas-liquid chromatography.     

Bismarck Brown As A Stain For Mucoproteins

Bismarck brown has been used for many years as a stain for mucin. But some types of mucoprotein are so water-labile that they cannot be demonstrated by the aqueous or weak alcoholic solutions usually employed. It has been found that Bismarck brown in slightly acidified, strong alcoholic, solution stains mucin. A simple method is given for using this solution for staining water-stable mucoproteins. Another method is included in which full precautions are given for avoidance of water at all stages subsequent to fixation. This method must be used for the more water-labile mucoproteins. By the use of these methods, it has been possible to demonstrate a wide range of mucoproteins including those of the mast cells of Hardie and that of the zona pellucida of the graafian follicle.     

Functional-morphologic changes in the gastric and duodenal mucosa during prolonged administration of prednisolone

A prolonged administration (up to 25 days) of prednisolone to rats (0.4 mg per 100 g of the body mass) reveals some functional-morphological changes in the secretory apparatus and mucin production in the stomach and the duodenum: an increased functional activity of parietal, principle, Ecl- and G-cells of the stomach, Ec-cells and goblet cells of the duodenum (except Ec-cells and goblet cells in the outlet part of the stomach), and by the end of the experiment--hyperplasia of the parietal, G-cells and goblet cells. A conclusion is made that the increased acido-peptic activity of the gastric juice and the erosive-ulcerous complications under a prolonged administration of prednisolone result from an increased functional activity of the fundal glands, G-, Ecl-cells and Ec-cells of the duodenum with a subsequent hyperplasia of the G-cells by the 25th day of the experiment, as well as an insufficient mucin formation in the mucous membrane of the stomach, especially in its outlet part as a consequence of a decreased functional activity of the Ec-cells.     

Evaluation of Polymer Mucoadhesiveness by the Use of Acoustic Spectroscopy

An innovative and simple methodology has been developed and used for the evaluation of mucoadhesive properties of several polymers by means of sound speed measurements using high-resolution acoustic spectroscopy. In systems made of polymers in water, variations in hydration shell of polymeric chains determine changes of dispersions compressibility, and this phenomenon can be monitored by sound speed measurements. Four different polymers have been selected, namely PEG 6000, Carbopol 974, HPMC K4M, and Pectin 200/USP, all characterised by very different mucoadhesive properties. Samples made of each polymer alone (0.3–1.0% w/w) or in mixture with mucin (mucin fixed at 1.0% w/w) in water were investigated while using high-resolution ultrasonic spectrometer at two different frequencies (5.2 and 8.2 MHz). Polymer–mucin interaction was evaluated comparing experimental sound speed values of polymer–mucin samples with their theoretical values derived from the addition of sound speeds obtained while analysing each component alone. Results demonstrated the ability of the acoustic method to discriminate between mucoadhesive and no mucoadhesive polymer–mucin dispersions and allowed also the comparison between their mucoadhesive strengths. The study has therefore demonstrated the potential of using high-resolution ultrasonic spectroscopy to evaluate the polymers’ mucoadhesiveness, with the great advantage of testing small amount of samples even if opaque.     

荧光酶标免疫分析在食品微生物检测中的应用

荧光酶标免疫分析是一种新型的快速检测微生物的方法,具有方便、快速、灵敏、准确等优点,已越来越多地被用于进出口食品中致病菌的检测。本文介绍了荧光酶标免疫分析法的原理、特点以及在食品微生物检测中的应用和未来发展方向。     

A chemical method for the deglycosylation of proteins

A simple and rapid chemical method for the deglycosylation of glycoproteins has been developed. The method involves the incubation of protein with trifluoromethanesulfonic acid at 0 degrees C from 0.5 to 2 h followed by the neutralization of the acid with aqueous pyridine at -20 degrees C. The method has been applied effectively to fetuin, ovine submaxillary mucin, ovine lutropin, and human choriogonadotropin. In 1 h almost all of N- and O-linked carbohydrates from ovine lutropin and human choriogonadotropin, with the exception of the linkage N-acetylglucosamine or N-acetylgalactosamine, were removed. Similarly, in 1 h all N-linked carbohydrates, excepting again the linkage sugar, in fetuin were degraded. Longer reaction times up to 2 h completely removed the O-linked carbohydrate chains from fetuin and ovine submaxillary mucin. The deglycosylated hormones thus prepared retained their immunological and biological activities.     

Dissociations within association cortex

The parietal lobes of nonhuman primates have been well characterized by single neuron recordings, yet clear partitioning of the human parietal lobes has not been clearly demonstrated. An elegant new fMRI study by Simon and colleagues (this issue of Neuron) suggests that parietal cortex does indeed contain dissociable subregions, each specialized for a particular visuomotor or cognitive function.     

Localisation of epidermal growth factor receptor in mucous cells of human salivary glands

EGFR activation has been related to an increase in synthesis and secretion of mucins in epithelial cells, so that the use of EGFR tyrosine kinase inhibitors has been proposed in the therapy of mucin hypersecretory diseases. In this paper, we describe the ultrastructural localisation of EGFR in the mucous elements of human major and minor salivary glands and relate it to mucin distribution. A post-embedding immunogold staining method has been applied to normal surgical samples of human submandibular, sublingual, and labial glands, using a mouse monoclonal antibody specific for the intracellular domain of human EGFR. In mucous cells of all the glands examined, specific reactivity was detected in the cytoplasmic basolateral portions and near the mucous droplets, but not on cell surfaces. Since this pattern of labelling must be related to the internalisation process of the ligand-GFR complex, our results support the hypothesis that EGFR activation takes place in mucous cells and affects mucin production in human salivary glands.     

Aggregation of mucin by chromium(III) complexes as revealed by electrokinetic and rheological studies: influence on the tryptic and O-glycanase digestion of mucin

In the present study, the impact of chromium(III) complexes ([Cr(salen)(H2O)2](+) (1), [Cr(en)3]3+ (2) and [Cr(EDTA)(H2O)]- (3)) on the biophysical properties of mucin like specific viscosity, zeta potential and particle size has been investigated. It is evident from the present investigation that the nature of the coordinated ligand has a major role to play in bringing about the changes in the physical characteristics of the glycoprotein. It was observed that (1) and (3) because of their coordinate mode of binding lead to decrease in the specific viscosity of mucin, whereas (2) on the other hand was found to bring about drastic increase in the mucin viscosity due to sol-gel transition in the mucin conformation. Complex (2) was found to gradually lower the zeta potential value of mucin (particle size=51.5 nm) from -24.8 +/- 1.31 mV to -0.58 +/- 0.30 mV, which reveals aggregation (particle size=216 nm) and subsequent sedimentation of mucin with an increase in the average diameter of mucin particles. The binding of (2) to mucin was found to impart resistance to mucin against both tryptic and O-glycanase digestion, suggesting that, the aggregation of mucin causes conformational as well as configurational changes in the glycoprotein; thus perturbing the location of carbohydrate domains.