Effects of calcium and bound sialic acid on the viscosity of mucin

A viscosity drop was observed when pig gastric mucin was titrated with HCl below pH 3.0. When EDTA was present, addition of HCl resulted in an increase in viscosity between pH 3.0 and pH 1.0. Previous treatment of the mucin solution with neuraminidase abolished the pH-dependent viscosity rise in the presence of EDTA.     

Studies on mineral ion absorption by plants

Summary The effect of Al and P on the growth of lucerne (Medicago sativa) was studied in nutrient solutions in which aluminium phosphate did not precipitate. Al and P retained in the free space of the roots was washed out with 0.1N HCl/O₄ at 5°C. The inhibitory effect of Al on growth was much less at pH 5 than at pH 4.5, although 3 to 4 times as much Al was found in the roots and shoots of the pH 5 plants.It is suggested that the low toxicity of high contents of Al was due to a portion of the uptake at pH 5 being in the form of polymeric aluminophosphate complexes of low net charge density. The optimum pH for the formation and polymerization of such complexes is around 5, and their composition depends on the P/Al mole ratio of the initial solutions. Washing³²P-labelled roots in unlabelled P solutions containing Ca showed that 12–43 per cent more of the total label diffused out of the Al-treated roots at pH 5 than from control roots. This was consistent with estimates by solution analysis of 16–36 per cent (depending on the P/Al mole ratio) of the P present in the original uptake solutions being complexed with Al.     

Bioadhesive properties of polygalacturonides against colonic epithelial membranes

Diseases of the gastrointestinal system are often related with irritations or pathological changes of mucous membranes. In an ex vivo system based on porcine colonic tissue various neutral and acidic polysaccharides were tested concerning their bioadhesive potential in order to form artificial mucin layers on colon epithelial membranes. Rhamnogalacturonans with a low degree of esterification and linear oligogalacturonids derived from pectin showed significant bioadhesion against colonic mucous membranes. In contrast highly esterified pectins and neutral polysaccharides were ineffective. Within a structure–activity relationship linear, strongly acidic homogalacturonides were shown to be most adhesive agents. Esterification, branching or non-linear backbone structures will reduce the adhesive properties. The bioadhesive effects were concentration-dependent. Polysaccharide layers, located exclusively on the apical membrane surface of colonic tissue, were visualized by fluorescent microscopy. The adhesion of the exogenous galacturonides on the tissue surface was mediated by interaction with the endogenous mucin, for the release of the endogenous mucines with a mucolytic agent resulted in a decreased bioadhesion of exogenous galacturonides. Additionally, mucin–galacturonide synergism was shown by rheological methods. The artificial mucin layers provide protective effects on colonic mucous membranes against toxic agents as shown by incubation of the tissue with TritonX-100.     

Enhancement of the viscosity of mucin by serum albumin.

The interaction of serum albumin with a model epithelial mucin from pig stomach was explored by rotary viscometry. During 30 min of incubation of human serum albumin(20mg/ml) and pig gastric mucin (8mg/ml) in iso-osmotic buffers at 37 degrees C, the solution became markedly viscous. Viscosity enhancement was proportional to albumin concentration (2-40mg/ml), was most pronounced under conditions of low shear rate (less than 45S-1), and was considerably greater than the additive or multiplicative viscosity values calculated from albumin or mucin solutions measured separately. The viscous mucin-albumin complex was destroyed by high shear rates (greater than 90S-1), but slowly re-formed under zero shear conditions. Elevation of pH (7 to 9), ionic strength (0.1 to 1.0), and addition of disodium EDTA (5mM) did not cause marked or specific alterations in the viscosity of the mixture, suggesting that electrostatic interactions probably do not stabilize mucin-albumin complexes. Urea (7M) and heating (35 to 55 degrees C) caused a major increase in the viscosity of mucin and mucin-albumin mixtures, suggesting that rupture of hydrogen bonds, unfolding and partial denaturation of mucin promotes greater intertangling (possibly hydrophobic interactions) between mucin and albumin molecules. The implications of mucin-albumin interaction in diseases associated with mucus obstruction are briefly discussed.     

pH-dependent conformational change of gastric mucin leads to sol-gel transition

We present dynamic light scattering (DLS) and hydrophobic dye-binding data in an effort to elucidate a molecular mechanism for the ability of gastric mucin to form a gel at low pH, which is crucial to the barrier function of gastric mucus. DLS measurements of dilute mucin solutions were not indicative of intermolecular association, yet there was a steady fall in the measured diffusion coefficient with decreasing pH, suggesting an apparent increase in size. Taken together with the observed rise in depolarized scattering ratio with decreasing pH, these results suggest that gastric mucin undergoes a conformational change from a random coil at pH >/= 4 to an anisotropic, extended conformation at pH < 4. The increased binding of mucin to hydrophobic fluorescent with decreasing pH indicates that the change to an extended conformation is accompanied by exposure of hydrophobic binding sites. In concentrated mucin solutions, the structure factor S(q, t) derived from DLS measurements changed from a stretched exponential decay at pH 7 to a power-law decay at pH 2, which is characteristic of a sol-gel transition. We propose that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated.     

Effect of pH on the association behavior in aqueous solutions of pig gastric mucin

In this study, dynamic light scattering (DLS), turbidity, and rheo-small angle light scattering (rheo-SALS) methods have been utilized to examine the impact of pH (1 < or = pH < or = 7) on aqueous solutions of noncommercial purified pig gastric mucin. The asymmetric flow field-flow fractionation (AFFFF) measurements established that the mucin sample has a high molecular weight and is polydisperse. DLS measurements on dilute solutions of mucin disclosed large interchain aggregates at pH 2, where the polymer has a low charge density or is uncharged. At lower or higher values of pH, mucin is charged and the tendency of forming interpolymer complexes is affected. In the semidilute concentration regime, pronounced junction zones ('lumps' of polymer) are evolved and a heterogeneous connected network is formed at pH 2, whereas the association structures are disintegrated (smaller 'lumps') at lower or higher pH values due to electrostatic repulsive interactions, and a more homogeneous network is evolved. The DLS and viscosity results at pH 1 indicate the development of a fragmented network, composed of contracted chains that are decorated by some positive charges. The effect of shear flow on the structure of semidilute solutions of mucin was investigated with the aid of rheo-SALS methods. The scattered intensity revealed a strong upturn at low values of the wave vector (q) for mucin solutions at pH 2 and pH 4, which suggests the evolution of large association domains. At these pH values, a flow-induced anisotropy in the 2D SALS patterns in the form of elliptical shapes was observed at high shear rates.     

Interacting effects of pH,aluminium and base cations on growth and mineral composition of the woodland grasses Bromus benekenii and Hordelymus europaeus

The influence of base cation concentrations on pH and aluminium sensitivity of the woodland grasses Bromus benekenii and Hordelymus europaeus was studied in flowing solution culture experiments. Plants were exposed to low pH (3.9, experiment 1) and Al concentrations of 19 and 37 M (experiment 2) at two base cation (Ca+Mg+K) levels, all within the ranges measured in natural forest soil solutions. Elevated base cation concentrations ameliorated both H and Al toxicity, as indicated by increased root and shoot growth. In the third experiment, interactions between pH (4.3 and 4.0) and Al (0 and 19 M) were investigated. It was shown that the combined toxicity effects of H and Al were not greater than the separate H or Al effects. Tissue concentrations of base cations and Al increased with increasing concentrations in the solution, but were also influenced by the base cation : Al ratio. Relating the experimental evidence with the composition of forest soil solutions suggests an important role of soil pH and Al in controlling the distribution of the two species. Growth conditions also differ at various soil depths. Concentrations of free cationic Al were higher and base cation concentrations lower at 5–10 cm than at 0–5 cm soil depth. Increasing base cation concentrations may protect roots from both H and Al injury during periods of drought when concentrations of most elements increase in the soil solution, whereas molar ratios between base cations, H and Al remain unchanged.     

Is the Ca:Al ratio superior to pH,Ca or Al concentrations of soils in accounting for the distribution of plants in deciduous forest?

The distributions of vascular plants in south Swedish deciduous forests were related to exchangeable (exc) and soil solution concentrations of H⁺ (pH), Ca, Al and the Ca:Al ratios within these fractions. Topsoils (0–5 cm) of 172 sites with a pH_KCl of 3.2–3.9 (corresponding to 3.7–4.4 in soil solution) were used. In the soil solution both total Al_t and quickly reacting Al_r were determined. Exchangeable concentrations were generally well related to plant distributions, the highest correlation coefficients usually being given by pH_KCl>Ca_exc>Al_exc.>(Ca:Al)_exc. The (Ca:Al)_exc ratio was clearly inferior. Out of the soil solution variables studied, Ca concentration, followed by pH, was best correlated with plant distributions, Al_t, Al_r, and the Ca:Al ratios having similar and lower coefficients. It is concluded that the use of Ca:Al ratios as a general measure of Al toxicity in controlling plant distributions is rather problematic. It seems difficult to apply evidence for Ca-Al interactions from solution culture experiments to field conditions when measured as exchangeable or soil solution concentrations of the soil.     

Effect of lipids on the structure and rheology of gels formed by canine submaxillary mucin

Rheological experiments have shown that canine submaxillary mucin (CSM) forms gels in aqueous solution at low ionic strength and in 6M GdnHCl. Examination of specimens of intact CSM and also its subunits prepared by reduction and carboxymethylation showed that the presence of lipid increases the gel-forming capability, probably as a result of enhancement of the intermolecular hydrophobic interactions. The rheological evidence for gelation is that substantially larger values of the oscillatory storage modulus, G'(ω), and the dynamic complex viscosity, η^*(ω), are observed for lipid-containing CSM. TMs is backed up by electron micrographs of freeze fractured specimens, where we observe a network morphology in which the cross-links are formed as a result of non-bonded interactions between a number of CSM chains. The intermolecular interactions responsible for gelation probably involve hydrophobic association between the interdigitated oligosaccharides, and/or between the non-glycosylated regions of the protein core, and can occur even in a highly chaotropic medium (6M GdnHCl). In contrast to previous experiments with porcine submaxillary mucin and human tracheobronchial mucin, which form microphase-separated gels in aqueous solution, CSM solutions undergo macroscopic phase separation into polymerrich (gel) and polymer-poor (sol) phases. These data point to stronger hydrophobic interactions in lipid-containing CSM.     

Effects of rabbit gastrointestinal mucins and dextran on hydrochloride diffusion in vitro

We compared a viscous fingering formation of hydrochloric acid (HCl) in rabbit corpus, antral and duodenal mucins and with dextran under neutral and acidic conditions with respect to relative viscosity, molecular mass, and carbohydrate composition. The effect of desialyzation of duodenal mucin on the viscous fingering formation of HCl was also examined. HCl (0.1 N) was injected into 1% solutions of mucins and dextran and a subsequent viscous fingering formation was assessed based on an influx volume rate of HCl. A low influx volume rate indicates a high ability of the solutions to produce viscous fingers. The influx volume rate of HCl was lowest in duodenal mucin followed bl corpus mucin, antral mucin, and dextran at pH 7. The influx volume rate of HCl was inversely correlated with the relative viscosity of the solution. Maximum molecular masses were large in the order of corpus, antral, and duodenal mucins, and they were larger than dextran T2000. Rabbit gastrointestinal mucins were very polydisperse system. Duodenal mucin contains more sialic acid than gastric mucins; the influx volume rate of HCl increased in desialylated duodenal mucin. It is suggested that the higher ability of gastric mucins to prevent HCl diffusion than dextran were due to the differences in the molecular mass. The ability of duodenal mucin to prevent HCl diffusion was probably attributed to its high sialic acid content, which may reflect a physiological role of duodenal mucin in the duodenum that has to deal with HCl influx from the stomach.     

Aluminium accumulation in root cell walls coincides with inhibition of root growth but not with inhibition of magnesium uptake in Norway spruce

High levels of aluminium in the soil solution of forest soils cause stress to forest trees. Within the soil profile, pH and aluminium concentration in the soil solution vary considerably with soil depth. pH strongly influences the speciation of A1 in solution, and is a factor when considering toxicity of A1 to roots. Norway spruce ( Picea abies [L.] Karst.) seedlings were grown for 7 weeks in nutrient solutions at pH 3.2, 4.0 or 5.0 containing 0, 100 or 400 µ M A1. At the end of this period, seedling growth, the cation exchange capacity of the roots and the amount of exchangeable Ca and Mg in roots were determined. A1 concentrations in whole roots, root segments, and in needles were measured. Using X‐ray microanalysis, the concentrations of Al, Ca, Mg and P were determined in cortical cell walls. We wanted to test the hypotheses that (1) the amount of Al bound to cation exchange sites can be used as a marker for Al toxicity and (2) the Mg concentration of needles is controlled by the amount of Mg bound to cation exchange sites. Low pH reduced the inhibition of Al on root growth and shoot length. Both low pH and Al lowered the concentration of Ca and Mg in needles. Al concentrations in the roots decreased as the pH decreased. In the roots, Al displaced Mg and Ca from binding sites at the root cortical cell walls. A comparison of the effects of Al at the different pH values on root growth and Mg concentration in the needles, suggests that, at pH 5.0, an Al fraction in the apoplast inhibits root growth, but does not affect Mg uptake. This fraction of Al is not available for transport to the shoots. In contrast, Mg uptake is strongly affected by Al at pH 3.2, although only very low levels of Al were detected in the roots. Thus, Al accumulation in the apoplast is a positive marker for Al effects on root growth, but not Mg uptake. The Mg concentration of needles is not controlled by the amount of Mg bound to cation exchange sites.     

Biosorption of Cr, Cu and Al bySargassum biomass

The biosorption and desorption of Cr, Cu and Al were carried out using brown marine algaeSargassum fluitans biomass, known as the good biosorbent of heavy metals. The content of alginate bound to light metals could be changed by physical and chemical pretreatment. The maximum uptake of Cr, Cu and Al was independent of the alginate content. The maximum uptake of Al was two times(mole basis) than those of Cu and Cr. The aluminum-alginate complex was found in the sorption solution of raw and protonated biomass. Most of Cu, Al and light metals sorbed in the biomass were eluted at pH 1.1. However, only 5 to 10% of Cr sorbed was eluted at pH 1.1. The stoichometric ion exchange between Cu and Ca ion was observed on Cu biosorption with Ca-loaded biomass. A part of Cr ion was bound to biomass as Cr(OH)₂ ⁺ or Cr(OH)²⁺. Al was also bound to biomass as multi-valence ion and interfered with the desorbed Ca ion. The behavior of rawS. fluitans in ten consecutive sorption-desorption cycles has been investigated in a packed bed flow-through-column during a continuous removal of copper from a 35 mg/L aqueous solution at pH 5. The eluant used was a 1%(w/v) CaCl/HCl solution at pH3.     

MEP chromatography of antibody and Fc-fusion protein using aqueous arginine solution

MEP HyperCel resin, one of the Protein-A mimetic columns, is designed to bind antibodies at physiological pH and elutes the bound antibodies at mildly acidic pH. We have tested aqueous arginine solution for washing and elution of the resin. To our surprise, bound antibody and Fc-fusion protein eluted at pH 7.0 using 1 M arginine solution. Various solvent additives were then examined at pH 7.0. Among the tested additives, urea and arginine were the only additives that were effective in elution. Thus, urea and arginine at low concentrations were effectively used for washing the resin. NaCl and MgCl₂ at 0.1–1 M and ethanol at 5–20% were not effective. Based on these observations, it appears that protein binds to MEP resin through both polar and hydrophobic interactions with some contribution of electrostatic interaction, which can be simultaneously reduced by arginine or urea. On the other hand, Mabsorbent, another Protein-A mimetic column, appears to be more non-specific and non-selective.     

Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins

Background

The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis.

Methods

The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence.

Results

The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucinShigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells.

Conclusions

Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells.     

Formation of spherical iron(III) oxyhydroxide nanoparticles sterically stabilized by chitosan in aqueous solutions

The interactions between the cationic polymer chitosan (Chit) and iron(III) were investigated. The solution properties were studied by pH-metry, viscometry and dynamic light scattering. Solid state iron(III)-Chit samples were also prepared and characterized by IR spectroscopy and electron microscopy. In aqueous solutions, the precipitation pH of the iron(III) oxyhydroxide (FeOOH) is significantly shifted towards the higher pH values in the presence of Chit indicating that some interaction takes place between the iron(III) and the polymer. However, the additivity of the pH-metric titration curves, the lack of variation both in the viscometric and IR spectra of Chit in the presence and absence of iron(III), indicate the lack of direct complexation between the Chit and ferric ions. Isolated FeOOH nanospheres of 5-10 nm diameter were observed on the transmission electron microscopic pictures of samples obtained from solutions containing iron(III) and Chit, while from DLS measurements hydrodynamic units with a few hundred nm in diameter were identified. Our data support that Chit acts as steric stabilizer and inhibits the macroscopic aggregation of the subcolloidal FeOOH particles. Thus the iron(III)-Chit interactions offer a simple and economic way to fabricate nanometric size FeOOH spheres, morphologically similar to the core of iron(III)-storage protein, ferritin.     

Viscosity study of interactions between sodium alginate and CTAB in dilute solutions at different pH values

Interactions between anionic polyelectrolyte sodium alginate and the cationic surfactant cetytrimethylammonium bromide (CTAB) have been investigated by viscosity measurement techniques. The polymer–surfactant interactions are observed between alginate and CTAB at different pH in dilute solution. The results show that the rheological response of alginate dilute solutions is sensitive to a change of pH in the low pH range. The steady shear and intrinsic viscosity measurements reveal that the strong association between alginate and CTAB by electrostatic attraction above pH 5.0. However, as the pH value of solution decrease from 5.0 to 3.0, the strong association between alginate and CTAB is affected by not only electrostatic attraction but also hydrophobic interaction.     

An M(III)-facilitated flocculation technique for enzyme recovery and concentration

An inexpensive and rapid flocculation/dissolution technique based upon Al(III) or Fe(III) was shown to be effective in recovering and concentrating up to 84% of the active enzyme from two dilute enzyme systems. The enzymes, a protease (Caldolysin) and a beta-glucosidase, were precipitated at constant pH and ambient temperature by addition of Fe(III) or Al(III) solutions. Resulting colloidal hydrous oxide particles bound the enzymes enabling subsequent separation from the media by low speed centrifugation. The enzymes were recovered from the protein/M(III) precipitate by complexing the M(III) with citrate. A concentration factor of 94 was obtained for the beta-glucosidase system when the initial concentration was less than 1 mg.ml-1.     

Rheological properties of extracellular polysaccharide, Pestan, produced by Pestalotiopsis sp.

Summary The fluid behaviour of Pestan produced from Pestalotiopsis sp. KCTC 8637P was as a non-Newtonian fluid. The rheological behaviour of Pestan solution was examined by Power-law model, Herschel-Bulkley model and Arrhenius equation. As the result, Pestan solution was pseudoplastic behaviour with yield stress. According to increase of Pestan concentration, its flow index was decreased. Thus, low concentrations of Pestan solution were well exposed pseudoplastic property. Apparent viscosity of 0.2 % Pestan solution was 268.2 cP at 14.3 sec^–1 and was higher about 2.8 times than that of Xanthan gum solution. Apparent viscosity of Pestan solution was stable over a wide pH and was maximum at pH 8. Also, consistency index of Pestan solution was very stable over wide temperature than that of Xanthan gum solution.     

Blood modeling using polystyrene microspheres

The steady flow viscosity at shear rates 0 to 120 sec-1 and dynamic viscoelasticity at frequencies 0.02 to 0.8 Hz were determined for aqueous suspensions of uniform polystyrene microspheres of 1.0 micron diameter. Rheological properties of the microsphere suspensions were Newtonian for particle concentrations up to 32%. By introducing dextran and calcium chloride into the particle suspensions, non-Newtonian behavior was produced similar to that observed for human blood. The cooperative effects of dextran and calcium ions promoted aggregation of particles at a concentration as low as 12%. Thus, a suspension of uniform sized spherical polystyrene particles in aqueous solution of dextran may be made to mimic blood by controlling the surface charge on the polystyrene spheres using addition of calcium ions to the medium.     

Properties of non-polymerizable tropomyosin obtained by carboxypeptidase A digestion.

Tropomyosin digested with carboxypeptidase A [EC 3.4.12.2] (CTM) shows a lower viscosity than the undigested protein in solution. From the relation between the viscosity decrease and the amount of amino acids liberated from the carboxyl terminus during this digestion, it is inferred that loss of the tri-peptide-Thr-Ser-Ile from the C-terminus is responsible for the decrease in viscosity. The secondary structure of -TM was not affected by the digestion according to circular dichroic measurements. The viscosity of CTM did not increase in methanol-water mixtures, whereas that of tropomyosin increased markedly. These results indicate that polymerizability was lost upon the removal of a small peptide from the C-terminus without change in the secondary structure. A decrease in the viscosity of tropomyosin solutions was observed on the addition of CTM, indicating that CTM interacts with intact tropomyosin. The dependence of the viscosity decrease on the amount of CTM showed that CTM binds tropomyosin in a one-to-one ratio as a result of end-to-end interaction. Since paracrystals having a 400 A repeated band structure could be grown in the presence of Mg ions at neutral pH, side-by-side interactions in CTM molecules remain intact, even though polymerizability is lost. The disc gel electrophoretic pattern showed that troponin could bind to CTM, but no increase in viscosity due to the complex was observed in solution. That is, the C-terminal part of tropomyosin is not required for the formation of the complex. The amount of CTM bound to F-actin was less than half of that bound to undigested tropomyosin, and could be reduced to one-tenth by a washing procedure. In the presence of troponin, however, the amount recovered to the level of tropomyosin normally bound to F-actin. Therefore, it is concluded that troponin is bound in the middle of the tropomyosin molecule and strengthens the binding of tropomyosin to F-actin.