Pig Gastric Mucus Research Articles

Enhanced microscopic dynamics in mucus gels under a mechanical load in the linear viscoelastic regime

Mucus is a biological gel covering the surface of several tissues and ensuring key biological functions, including as a protective barrier against dehydration, pathogen penetration, or gastric acids. Mucus biological functioning requires a finely tuned balance between solid-like and fluid-like mechanical response, ensured by reversible bonds between mucins, the glycoproteins that form the gel. In living organisms, mucus is subject to various kinds of mechanical stresses, e.g., due to osmosis, bacterial penetration, coughing, and gastric peristalsis. However, our knowledge of the effects of stress on mucus is still rudimentary and mostly limited to macroscopic rheological measurements, with no insight into the relevant microscopic mechanisms. Here, we run mechanical tests simultaneously to measurements of the microscopic dynamics of pig gastric mucus. Strikingly, we find that a modest shear stress, within the macroscopic rheological linear regime, dramatically enhances mucus reorganization at the microscopic level, as signaled by a transient acceleration of the microscopic dynamics, by up to 2 orders of magnitude. We rationalize these findings by proposing a simple, yet general, model for the dynamics of physical gels under strain and validate its assumptions through numerical simulations of spring networks. These results shed light on the rearrangement dynamics of mucus at the microscopic scale, with potential implications in phenomena ranging from mucus clearance to bacterial and drug penetration.

Effect of pH on the viscoelastic properties of pig gastric mucus

Mucus is a biomaterial with peculiar, gel-like viscoelastic properties, and bearing different functionalities, depending on the different mucosae it covers. It is clear that these functionalities have to stay effective throughout the in vivo broad range of physiological pH values at which the mucus is exposed. We sought here to determine the effect of pH on the rheological properties of ex vivo mucus. We demonstrate that viscoelastic properties of gastric mucus are quite “stable” to pH changes, in marked contrast with the pH sensitivity of purified mucin gels. We also find that the rheological features of porcine gastric mucus are reversible when the system is first alkalized up to solubilization (pH > 8.5) and then re-acidified to its initial pH value.

Chitosan composite microparticles: A promising gastroadhesive system for taxifolin

Taxifolin possesses gastroprotective property but is characterized by low water solubility, is instabile in alkaline medium, and is degraded by the intestinal bacteria flora. The purpose of the work was therefore to produce a gastroadhesive formulation to prolong taxifolin residence time and release in the stomach. We first demonstrated that taxifolin is stable in simulated gastric fluid with or without pepsin and mucus, and is able to cross pig gastric mucus layer and stomach mucosa. Next, gastromucoadhesive microparticles composed of Syloid® AL-1 mesoporous silica, chitosan and HPMC were produced using spray-drying. Microparticles were characterized by a spherical shape and a mean volume-equivalent diameter around 12 μm. The optimized microparticles were able to release taxifolin and to adhere to pig stomach mucosa for 5 h.

Helicobacter suis infection alters glycosylation and decreases the pathogen growth inhibiting effect and binding avidity of gastric mucins

Helicobacter suis is the most prevalent non-Helicobacter pylori Helicobacter species in the human stomach and is associated with chronic gastritis, peptic ulcer disease, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. H. suis colonizes the gastric mucosa of 60–95% of pigs at slaughter age, and is associated with chronic gastritis, decreased weight gain, and ulcers. Here, we show that experimental H. suis infection changes the mucin composition and glycosylation, decreasing the amount of H. suis-binding glycan structures in the pig gastric mucus niche. Similarly, the H. suis-binding ability of mucins from H. pylori-infected humans is lower than that of noninfected individuals. Furthermore, the H. suis growth-inhibiting effect of mucins from both noninfected humans and pigs is replaced by a growth-enhancing effect by mucins from infected individuals/pigs. Thus, Helicobacter spp. infections impair the mucus barrier by decreasing the H. suis-binding ability of the mucins and by decreasing the antiprolific activity that mucins can have on H. suis. Inhibition of these mucus-based defenses creates a more stable and inhabitable niche for H. suis. This is likely of importance for long-term colonization and outcome of infection, and reversing these impairments may have therapeutic benefits.

Mucus as an Arrested Phase Separation Gel

We investigate the spontaneous restructuring and aging behavior of pig gastric mucus gels by combining rheology and nonconventional static and dynamic light scattering. We find that the mucus elasticity weakens over time, concomitantly to structural evolution toward a locally more compact configuration and the onset of large scale density fluctuations. We interpret this behavior as stemming from an arrested phase separation, as observed in many gel-forming polymer systems. This scenario is confirmed by space- and time-resolved dynamic light scattering data, showing that the gel restructuring is due to intermittent bursts of spatially correlated rearrangements, leading to ballistic dynamics.

The Effect of Some Preservatives Used in Nasal Preparations on the Mucus and Ciliary Components of Mucociliary Clearance

Efficient mucociliary clearance is a function of the physical properties of the mucus coupled to appropriately functioning cilia and may be altered by substances affecting ciliary beat frequency (CBF). Therefore the effect of preservatives on CBF was investigated using a photoelectronic technique. Methyl-p-hydroxybenzoate, propyl-p-hydroxybenzoate, chlorbutol and chlorocresol inhibited beat frequency, an effect which was reversible upon rinsing out the first three compounds but not chlorocresol. The effect of chlorhexidine and phenylmercuric borate on CBF was complicated by an interaction with chloride ions in the media used. EDTA did not appear to be ciliotoxic, while the effect of benzalkonium chloride was variable. Thiomersal halted ciliary beating after 40-100 min. Mucociliary clearance may also be affected by an alteration of the physical properties of the mucus layer, therefore the effect of each compound on the rheological properties of purified pig gastric mucus glycoprotein was investigated. None of the preservatives significantly altered the viscoelastic properties of the gel, measured using dynamic techniques.

Counter-ion binding to mucus glycoproteins.

The affinity of pig gastric mucus glycoprotein for counter ions of various valencies was examined. Ions of higher valency were bound with increasing avidity, but the degree of binding was apparently not influenced by the ionic radius. The affinity of the glycoprotein for the ions studied may be ranked: Fe3+ greater than Al3+ greater than Ca2+ greater than Cs+ congruent to Na+. The binding of calcium was inhibited by high ionic strength and was sensitive to the pH of the environment. Enzymatic removal of sialic acid slightly reduced the calcium binding capacity.

Evaluation of the bioadhesive property of Grewia gum in indomethacin tablet formulation in pig gastric mucus

The bioadhesive property of grewia gum for sustained release of Indomethacin from tablets was evaluated using pig gastric mucus as substrate. The tablets formulated by wet granulation contained 75 mg of the drug and 15 or 20 %w/w of the gum. Similar tablets made differently with carbopol 934, tragacanth and sodium carboxymethylcellulose (SCMC) were used as basis for comparison. Hydration of tablets and substrate surfaces was done using 0.1N hydrochloric acid and phosphate buffer providing pH values of 1.2 and 7.4 respectively. The force required to detach the tablets from substrate surface was determined in a testometric material testing machine. Drug release from polymer matrix tablets was fastest for tragacanth and slowest for carbopol 934. The detachment force of tablets from substrate was found to increase in the order: carbopol 934 > SCMC > grewia gum > tragacanth. The detachment force was affected by the pH of the hydration medium. On the basis of these grewia gum may find application as bioadhesive polymer for sustained release tablet formulation. Keywords: Indomethacin; Pig gastric mucus, Grewia gum, Bioadhesive performance Journal of Pharmacy and Bioresources Vol. 3 (2) 2006: pp. 62-69

Mucous Systems Show a Novel Mechanical Response to Applied Deformation

Mucous secretions have a wide range of biological functions that are intimately linked with their rheological properties. In addition, many mucous secretions are exposed to significant stress and deformation during physiological function. This study has examined the rheological response of three mucous systems, native pig gastric mucus, purified mucin gels, and mucin alginate gels, to increasing applied stress to a level sufficient to induce flow behavior. A novel, frequency-dependent stress hardening was observed in all three systems. This hardening behavior may play a significant role in the ability of mucous systems to resist mechanical disruption in the physiological state.

The gel matrix of gastric mucus is maintained by a complex interplay of transient and nontransient associations.

The gel nature of mucus is fundamental to its physiological functions; however, the structure of the mucus gel matrix is unclear. Here, small and large deformation rheology has been used to investigate the physical nature of the gel matrix and the forces that maintain this matrix in pig gastric mucus. The gelation process in mucus has been shown to be comparable with that of other polymer gel systems. Nongelling portions of mucin have been identified within the gel network, and the importance of transient, relaxable interactions to the maintenance of the mucus gel matrix has been demonstrated. The structure of the mucus gel matrix is considered in relation to the functional properties of mucus gels.

Disulfide-Bound Proteolytic Fragments of Gastric Mucin Are 100- and 140-kDa Proteins

Pig gastric mucus was tested for its autodegradative proteolytic degradation at pH 7.0, in the presence or absence of proteinase inhibitors and SDS. Samples of crude mucus were incubated at room temperature for 48 and 96 h in sodium azide stabilized buffer, pH 7.0, and urea-extracted mucin was purified. Electrophoretically homogenic mucin preparation was reduced and alkylated with iodo[14C]acetamide, and analyzed for labeled products. On 7.5% SDS/PAGE protein bands at 80 and 120 kDa were noted, but radioactivity was incorporated into 100- and 140-kDa bands, with increasing intensity from T0 to T96, and into high molecular mass mucin subunits. The results confirmed the autodegradative properties of gastric mucin and demonstrated that the 100- and 140-kDa fragments are the main proteolytical products of pig gastric mucin and are disulfide bound with the rest of the molecule.

Mucus glycoproteins from pig gastric mucosa: identification ofdifferent mucin populations from the surface epithelium.

Pig gastric mucins were isolated from the surface epithelium of the cardiac region, corpus and antrum using density-gradient centrifugation after extraction in 6 M guanidinium chloride. In CsCl/0.5 M guanidinium chloride, mucins solubilized from the cardiac region appeared as a broad unimodal band at 1.52 g/ml whereas those from the corpus and antrum occurred as high- and low-density populations at 1.50 and 1.45 g/ml respectively. High-iron diamine reacted more strongly with the cardiac mucins and the high-density populations from corpus and antrum than with the two low-density ones. In keeping with this, approx. 60% of the oligosaccharides from the former mucins and 20% from the latter contained sulphate. All surface epithelial cells of the cardiac region stained with high-iron diamine, whereas in the corpus only the epithelium in the bottom of the pits reacted, suggesting that the high-density population from this region originates from these cells. Mucins from all regions were composed of subunits, each containing highly glycosylated domains. The mucins from the cardiac region were larger than those from the corpus and antrum, and reduced subunits as well as high-molecular-mass glycopeptides from the cardiac mucins were larger than the corresponding fragments from the other regions. Ion-exchange HPLC showed that reduced subunits from the cardiac mucins and the high-density populations from the corpus and antrum were more 'acidic' than reduced subunits from the two low-density ones. All mucins contained a 'neutral'fraction, in particular those from the antrum. Pig gastric mucus thus contains a number of distinctly different mucin populations varying in buoyant density, size, 'acidity', glycosylation, sulphation and tissue origin.

Gastric mucus of the guinea pig: proton carrier and diffusion barrier.

Proton transport with the gastric mucus was investigated in the guinea pig in vitro by use of three experimental series. In series I, pH profiles were obtained in the mucus and mucosa of a gastric explant with fine-tipped double-barreled microelectrodes. With a luminal pH of 1.8, pH increased across this layer to approximately 6 at the epithelial surface. Thickness of the gastric mucous gel layer increased continuously by 170 +/- 100 microns/h in the unstimulated and by 450 +/- 120 microns/h in the histamine-stimulated preparation (means +/- SD). In series II, fresh guinea pig gastric mucus was obtained from the gastric mucosa and titrated at 10 degrees C from pH 6.5 to 0.7, followed by an incubation period of 30 min at 37 degrees C. During this incubation period, a spontaneous acidic shift was observed, corresponding to a proton release from the mucus of 130 +/- 19 mM. This proton release could be blocked by the pepsin inhibitor pepstatin and was not observed when titrating down to only pH 3. Buffer values calculated as the mean slope of the titration curves in the pH range of 7 to 3 averaged 40 mM/pH unit. In series III, when titration was repeated with purified porcine mucin, no proton release was observed during incubation at pH 1.0, unless pepsinogen (375 U/ml) had been added before titration. Proton release during incubation at pH 1.0 and 37 degrees C in the presence of pepsinogen averaged 50 mM. The data suggest that protons secreted by the gastric mucosa are buffered by the continuously secreted mucus and transported, bound to the proteins of the mucus, toward the gastric lumen. During this transport, pepsinogen is converted within the mucus to pepsin. Pepsin modifies the buffering properties of the mucus, whereby protons are released from the protein binding. Thus the mucus forms a vehicle for proton transport toward the gastric lumen while, at the same time, constituting a diffusion barrier to prevent proton backdiffusion toward the gastric epithelium.

Drug Diffusion through Cystic Fibrotic Mucus: Steady-State Permeation, Rheologic Properties, and Glycoprotein Morphology

One manifestation of cystic fibrosis (CF) is the presence of a viscid mucus secretion in the lungs. The clearance of this mucus is significantly slower than in “normals” due to uncoordinated beating of the cilia and the increased viscosity of the mucus. In these studies, the permeabilities of p-aminosalicylic acid, isoniazid, and pyrazinamide through unpurified CF respiratory mucus and through purified pig gastric mucus solutions were compared in order to evaluate the relative barrier properties of these mucus solutions. These model compounds, while not often used clinically in CF, are used in other pulmonary diseases and have the potential to be administered by inhalation delivery systems. Permeability studies were carried out in Side-Bi-Side diffusion cells fitted with a custom membrane holder capable of retaining the mucus solutions. Permeabilities through CF mucus solution and its fractions were compared to those measured through buffer and reconstituted purified pig gastric mucus. There were 28–75% decreases in drug permeability when pig gastric mucus was replaced by different CF mucus solutions. This indicates that optimal drug delivery directly to the lungs must take into account the decreased drug transport rate across diseased mucus in addition to drug loss due to drug binding to the glycoproteins or inefficient delivery via aerosolization. Transmission electron microscopy revealed minor differences in the glycoprotein strand structure between reconstituted pig gastric mucus and CF mucus primarily with regard to glycoprotein chain length and extent of branching. Similar viscoelastic behaviors between the CF gel fraction and synthetic CF mucus were observed. This model CF mucus system can simulate diseased mucus and can be utilized for in vitro studies to optimize drug permeability.

A glycosulphatase that removes sulphate from mucus glycoprotein.

A novel glycosulphatase has been purified from a mucus glycopeptide-degrading Prevotella from the colon. The purified enzyme removed inorganic [35S]sulphate from 35S-labelled native rat gastric mucus glycoprotein. Desulphation of mucus glycoprotein was initially rapid (19% complete after 10 min) but then plateaued, reaching only 33% after 3 h. Crude periplasmic extracts could remove 79% of the radioactivity as inorganic sulphate. These results suggest that steric hindrance may limit the access of the purified glycosulphatase to the mucus glycoprotein oligosaccharide chains in the absence of glycosidases, and/or that the enzyme may have the wrong specificity for some of the remaining sulphated sugars in the chains. The apparent molecular mass of the enzyme was 111 kDa as judged from gel exclusion chromatography, and it appeared to be composed of two identical subunits. The enzyme was localized in the periplasm of the bacterium, and using pig gastric mucus glycopeptide as a growth substrate markedly increased enzyme levels. Enzymic activity increased at the end of the growth phase. The substrate specificity of the enzyme was tested against low-molecular-mass sulphated molecules. The monosaccharides glucose 6-sulphate and N-acetylglucosamine 6-sulphate were rapidly desulphated, the latter being the major sulphated sugar in some mucus glycoproteins. Lactose 6-sulphate, galactose 6-sulphate, sulphated steroids and unsaturated disaccharide sulphate breakdown products from chondroitin sulphate were not desulphated. Glycosulphatases which can remove sulphate from mucus glycoproteins may play an important role in the degradation of highly sulphated mucus glycoproteins in the digestive tract, and could modify the effectiveness of mucus glycoproteins in mucosal protection.

Molecular fractionation of gastric mucus component by high-performance liquid chromatography: application to pig gastric mucus in vitro and to human gastric mucus collected by aspiration during gastric endoscopy

The glycoprotein molecular composition of antral and fundic adherent mucus has been studied by high-performance liquid chromatography on a silica gel column. Preliminary assays with pig gastric mucus allowed us to demonstrate the reproducibility of the method. The mucolytic activity of pepsin on this mucus demonstrates its ability to detect degradation of its glycoprotein components. This method was applied to control the state of pig antral mucosa that has previously been used in an in vitro antacid evaluation procedure, and also to study human fundic and antral mucus collected by aspiration from normal and diseased stomachs during upper gastrointestinal endoscopy. Different elution profiles were obtained with these samples, depending on the presence of non-degraded or degraded mucus or due to the lack of mucus on the mucosa.

Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

A comparison of the results of sequential hydrazinolysis-nitrosation and alkali-mediated cleavage-nitrosation of the O-linked oligosaccharides of gastric mucus glycoproteins

Analysis of the oligosaccharides released from pig gastric mucus glycopolypeptides by hydrazinolysis showed that degradation had occurred. Nitrosation of the products followed by reduction gave a mixture that had a low content of 2,5-anhydro- d-talitol, which implied destruction of much of the terminal reducing 2-amino-2-deoxy- d-galactose. Under the conditions of hydrazinolysis, cellobiose was largely unchanged but laminaribiose gave a complex mixture that probably contained glucose hydrazone ( 13C-n.m.r. data). In order to avoid degradation, the hydrazinolysis-nitrosation sequence should be applied to the reduced oligosaccharides released on cleavage with alkali.

On the interaction in solution of a candidate mucoadhesive polymer, diethylaminoethyl-dextran, with pig gastric mucus glycoprotein

Fluorescence Fig. 1. Histogram of spot fluorescent signal; artefacts have been distingiiished from spots as described in the text (u) For six fibroblasts treated with Dil-labelled LDL. The continuous line is the best fit of the log-normal distribution found in (h) for single particles, convolved to give up to five particles per spot. (h) For DiI-LDL on a polylysine-coated slide. The continuous line is the best fit of a log-normal distribution.

Effect of pepsin on partially purified pig gastric mucus and purified mucin

Partially purified native-pig gastric mucus and purified pig gastric mucin, prepared by column chromatography and caesium chloride (CsCl) density-gradient ultracentrifugation, were subjected to pepsin digestion. The products of peptic digestion were chromatographed on Sepharose CL-2B, and fractions were assayed for carbohydrate by the periodic acid-Schiff reaction. The polymeric gastric mucin in the purified mucin samples was readily degraded by pepsin. In sharp contrast, the polymeric mucin in the partially purified mucus was relatively resistant to pepsin digestion. In 45 min, pepsin degraded 40% of the polymeric mucin in the purified samples, whereas it produced no significant degradation (less than 10%) in the partially purified mucus samples. In partially purified gastric mucus, treated with CsCl but not fractionated by ultracentrifugation, digestion with pepsin was also slow and incomplete. This showed that differences in susceptibility between partially purified and purified preparations are not due to the chaotropic effects of CsCl. In addition, the recombination of low-density nonmucin fractions in CsCl ultracentrifugation with the mucin also resisted pepsin digestion. Finally, we have shown that the low-density fractions in mucus exhibited a strong inhibitory effect of peptic activity in vitro. We conclude that under our experimental conditions, pepsin has little effect on partially purified mucus, and our findings indicate an inhibitor of peptic digestion is present in native gastric mucus. It is likely, but unproven, that this inhibitor is a noncovalently bound lipid present in the low-density fraction.