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Microscale propulsion impacts a diverse array of fields ranging from biology and ecology to health applications, such as infection, fertility, drug delivery, and microsurgery. However, propulsion in such viscous drag-dominated fluid environments is highly constrained, with time-reversal and geometric symmetries ruling out entire classes of propulsion. Here, we report the spontaneous symmetry-breaking propulsion of rotating spherical microparticles within non-Newtonian fluids. While symmetry analysis suggests that propulsion is not possible along the fore-aft directions, we demonstrate the existence of two equal and opposite propulsion states along the sphere's rotation axis. We propose and experimentally corroborate a propulsion mechanism for these spherical microparticles, the simplest microswimmers to date, arising from nonlinear viscoelastic effects in rotating flows similar to the rod-climbing effect. Similar possibilities of spontaneous symmetry-breaking could be used to circumvent other restrictions on propulsion, revising notions of microrobotic design and control, drug delivery, microscale pumping, and locomotion of microorganisms.

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation., Competing Interests: Declarations of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Low grade appendiceal mucinous neoplasm (LAMN) is the primary source of pseudomyxoma peritonei (PMP). PMP may develop after seemingly complete resection of primary tumor by appendectomy, which is unpredictable due to lack of reliable prognostic indicators. We retrospectively reviewed 154 surgically resected LAMNs to explore if any of the macroscopic and microscopic characteristics may be associated with increasing risk of PMP development. Our major findings include: (1) As compared to those without PMP, the cases that developed PMP were more frequent to have (a) smaller luminal diameter (<1 cm) and thicker wall, separate mucin aggregations, and microscopic perforation/rupture, all suggestive of luminal mucin leakage; (b) microscopic acellular mucin presenting on serosal surface and not being confined to mucosa; and (c) neoplastic epithelium dissecting outward beyond mucosa, however, with similar frequency of neoplastic cells being present in muscularis propria. (2) Involvement of neoplastic cells or/and acellular mucin at surgical margin did not necessarily lead to tumor recurrence or subsequent PMP, and clear margin did not absolutely prevent PMP development. (3) Coexisting diverticulum, resulted from neoplastic or non-neoplastic mucosa being herniated through muscle-lacking vascular hiatus of appendiceal wall, was seen in a quarter of LAMN cases, regardless of PMP. The diverticular portion of tumor involvement was often the weakest point where rupture occurred. In conclusion, proper evaluation of surgical specimens with search for mucin and neoplastic cells on serosa and for microscopic perforation, which are of prognostic significance, should be emphasized., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In order to characterize the adhesion and deformation behavior between chitosan-modified liposomes and the mucin layer of the small intestine, mucin was coated on hydrophobic surface-modified carbon nanotube (CNT) probe of an atomic force microscope. The interaction between this mucin layer and the liposomes with or without chitosan modification in phosphoric acid buffer solution was determined by atomic force microscopy. The pH of the buffer solution was controlled at 2.8 and 7.0. The chitosan modification increased the attractive force between the liposomes and mucin layer during the separation process under both pH conditions. This result corresponded with that from a previous study about the liposome adhesion behavior on the surface of the small intestine of rats. By using the mucin-coated CNT probe, the long range and different types of attractive forces between the chitosan-modified liposomes and mucin layer was observed. Furthermore, the small-scaled deformation behavior change on the liposomal surfaces due to chitosan modification was also observed by the CNT probe. The detail deformation and adhesion behavior of the liposomes with or without chitosan modification was detected.

Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.

Mucus, with its burden of inspired particulates and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be "watery" and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ~250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate (KS), a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed KS-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190-1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, whereas the smaller adenoassociated virus penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies.

Moist mucosal epithelial interfaces that are exposed to external environments are dominated by sugar epitopes, some of which (e.g., sialic acids) are involved in host defense. In this study, we determined the abundance and distribution of two sialic acids to assess differences in their availability to an exogenous probe in isolated mucins and mucous gels. We used atomic force microscopy to obtain force maps of human preocular mucous and purified ocular mucins by probing and locating the interactions between tip-tethered lectins Maackia amurensis and Sambucus nigra and their respective receptors, α-2,3 and α-2,6 N-acetylneuraminic (sialic) acids. The rupture force distributions were not affected by neighboring sugar-bearing molecules. Energy contours for both lectin-sugar bonds were fitted to a two-barrier model, suggesting a conformational change before dissociation. In contrast to data from purified mucin molecules, the preocular gels presented numerous large clusters (19,000 ± 4000 nm(2)) of α-2,6 sialic acids, but very few small clusters (2000 ± 500 nm(2)) of α-2,3 epitopes. This indicates that mucins, which are rich in α-2,3 sialic acids, are only partially exposed at the surface of the mucous gel. Microorganisms that recognize α-2,3 sialic acids will encounter only isolated ligands, and the adhesion of other microorganisms will be enhanced by large islands of neighboring α-2,6 sialic acids. We have unveiled an additional level of mucosal surface heterogeneity, specifically in the distribution of pro- and antiadhesive sialic acids that protect underlying epithelia from viruses and bacteria., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The mechanisms by which mucus helps prevent viruses from infecting mucosal surfaces are not well understood. We engineered non-mucoadhesive nanoparticles of various sizes and used them as probes to determine the spacing between mucin fibers (pore sizes) in fresh undiluted human cervicovaginal mucus (CVM) obtained from volunteers with healthy vaginal microflora. We found that most pores in CVM have diameters significantly larger than human viruses (average pore size 340 +/- 70 nm; range approximately 50-1800 nm). This mesh structure is substantially more open than the 15-100-nm spacing expected assuming mucus consists primarily of a random array of individual mucin fibers. Addition of a nonionic detergent to CVM caused the average pore size to decrease to 130 +/- 50 nm. This suggests hydrophobic interactions between lipid-coated "naked" protein regions on mucins normally cause mucin fibers to self-condense and/or bundle with other fibers, creating mucin "cables" at least three times thicker than individual mucin fibers. Although the native mesh structure is not tight enough to trap most viruses, we found that herpes simplex virus (approximately 180 nm) was strongly trapped in CVM, moving at least 8,000-fold slower than non-mucoadhesive 200-nm nanoparticles. This work provides an accurate measurement of the pore structure of fresh, hydrated ex vivo CVM and demonstrates that mucoadhesion, rather than steric obstruction, may be a critical protective mechanism against a major sexually transmitted virus and perhaps other viruses.

In recent years, the exposure of biological systems to various nanomaterials has become an issue of great public concern. Although living organisms have arrays of biological defense mechanisms against exposure to exogenous compounds, the biochemical mechanisms allowing various nanomaterials to enter the body are not well understood. A unique example of a typical mucosal glycoprotein capable of binding and solubilizing nanomaterials in physiological solution is provided, suggesting a possible route for entry into biological systems.

Purpose: To evaluate human corneal endothelial mucin layer thickness and ultrastructure after phacoemulsification and irrigation-aspiration with either next generation ophthalmic irrigating solution (NGOIS) or BSS PLUS., Methods: Paired human corneas were mounted in an artificial anterior chamber, exposed to 3 minutes of continuous ultrasound (US) at 80% power using the Alcon SERIES 20000 LEGACY surgical system (n = 9) or to 2 minutes of pulsed US at 50% power, 50% of the time at 20 pps using the Alcon INFINITI Vision System (n = 5), and irrigated with 250 mL of either NGOIS or BSS PLUS. A control group of paired corneas did not undergo phacoemulsification or irrigation-aspiration (n = 5). Corneas were divided and fixed for mucin staining or transmission electron microscopy. Mucin layer thickness was measured on the transmission electron microscopy prints., Results: The mucin layer thickness in the continuous phaco group was 0.77 +/- 0.02 microm (mean +/- SE) with NGOIS and 0.51 +/- 0.01 microm with BSS PLUS (t test, P < 0.001). The mucin layer thickness in the pulsed phaco group was 0.79 +/- 0.02 microm with NGOIS and 0.54 +/- 0.01 microm with BSS PLUS (P < 0.001). The mucin layer thickness in the untreated control group was 0.72 +/- 0.02 microm. The endothelial ultrastructure was normal in all corneas., Conclusions: In this in vitro corneal model, NGOIS, due to its lower surface tension and higher viscosity, preserved endothelial mucin layer thickness better than BSS PLUS with both the INFINITI Vision System (pulsed US) and the LEGACY surgical system (continuous US).

A novel thermo- and pH-sensitive nanogel particle, which is a core-shell structured particle with a poly(N-isopropylacrylamide) (p(NIPAAm)) hydrogel core and a poly(ethylene glycol) monomethacrylate grafted poly(methacrylic acid) (p(MMA-g-EG)) shell, is of interest as a vehicle for the controlled release of peptide drugs. The interactions between such nanogel particles and artificial mucin layers during both approach and separation were successfully measured by using colloid probe atomic force microscopy (AFM) under various compression forces, scan velocities, and pH values. While the magnitudes of the compression forces and scan velocities did not affect the interactions during the approach process, the adhesive force during the separation process increased with these parameters. The pH values significantly influenced the interactions between the nanogel particles and a mucin layer. A large steric repulsive force and a long-range adhesive force were measured at neutral pH due to the swollen p(MMA-g-EG) shell. On the other hand, at low pH values, the steric repulsive force disappeared and a short-range adhesive force was detected, which resulted from the collapse of the shell layer. The nanogel particles possessed a pH response that was sufficient to protect the incorporated peptide drug under the harsh acidic conditions in the stomach and to effectively adhere to the mucin layer of the small intestine, where the pH is neutral. The relationships among the nanogel particle-mucin layer interactions, pH conditions, scan velocities, and compression forces were systemically investigated and discussed.

Carmine has been used in biological staining to demonstrate selectively nuclei, chromosomes or mucins, depending on the formulation. Throughout its history in science, complaints and frustrations have been expressed about dye quality. Inconsistencies in dye quality or identity have prevented thorough understanding of staining mechanisms and have caused many stain solutions to behave unsatisfactorily. The aim of this review is to (1) detail causes of these problems, which are rooted in history, geography and production, (2) offer ways to minimize problems and (3) provide modern explanations for stain behavior. Carmine is a "semi-synthetic" dye, i.e., a complex of aluminum and the natural dye cochineal (carminic acid). Carmine shows considerable batch-to-batch variability. Geography, politics, history, agricultural practices and iconography all contribute to the variability of cochineal. In addition, widely divergent manufacturing methods are used to produce carmine. Also, confusion in terminology has led to mislabeling. Pressure from the food industry for a more satisfactory colorant for acidic foods led to the introduction of a new dye, aminocarminic acid, which could enter the biological market inadvertantly. Improved methods of analysis should help the certification process by the Biological Stain Commission. Further standardization could be achieved by replacing most of the methods of solubilizing carmine. The majority of these methods use heat, which is likely to damage the dye molecule. Fortunately, carmine is readily dissolved by raising the pH of the aqueous solvent above 12, and a new form of the dye, now available commercially, is soluble in water without the need for heat or pH adjustment. Chemical structures and physical properties of carminic acid, carmine, aminocarminic acid and kermesic acid are reviewed. A new configuration for carmine is proposed, as well as possible changes to carminic acid and carmine molecules as a result of decomposition caused by heating. Each of the major classes of carmine-based stains is described as are possible mechanisms of attachment to specific substrates. Glycogen binds carmine through hydrogen bonding, and it is here that carmine decomposed by heat could have the greatest detrimental impact. Nuclei and chromosomes are stained via coordination bonds, perhaps supplemented by hydrogen bonds. Finally, acidic mucins react ionically with carmine. Specificity in the latter case may be due to unique polymeric carmine molecules that form in the presence of aluminum chloride.

CA125 antigen is a high molecular mass, structurally heterogeneous, mucin-type molecule expressed during embryonic development as well as in adult human tissues. This study was aimed at investigating its mucin-related property of ferning, as a general complementary way of characterization. Pregnancy-associated CA125 antigen (pCA125) was examined using light, transmission and scanning electron microscopy and compared with cancer-derived CA125 antigen (cCA125). The results obtained for spread-out, air-dried pCA125 and cCA125 samples revealed clear differences in the patterns of crystalline as well as amorphous material. Thus, the fern-like crystals were mainly sparsely distributed and their morphology was atypical. The extent of crystallization of pCA125 was moderately lower than that of cCA125 antigen, whereas variation in the size and spatial organization of fern crystals was evident. Besides the material with a crystalline appearance, differences in the organic substrate were also noticeable. In contrast to the sponge-like appearance of pCA125, cCA125 had a more compact structure. These initial data may be relevant for relating biochemical properties of CA125 antigen with its morphology as a basis for elucidating its still obscure function under different physiological conditions.

Atomic force microscopy (AFM) has been used to show that human ocular mucins contain at least three distinct polymer conformations, separable by isopycnic density gradient centrifugation. In this work we have used affinity purification against the anti(mucin peptide core) monoclonal antibody 45M1 to isolate MUC5AC gene products, a major component of human ocular mucins. AFM images confirm that the affinity-purified polymers adopt distinct conformations that coidentify with two of those observed in the parent population, and further reveal that these two different conformations can be present within the same polymer. AFM images of the complexes formed after incubation of 45M1 with the parent sample reveal different rates of binding to the two MUC5AC polymer types. The variability of gene products within a mucin population was revealed by analyzing the height distributions along the polymer contour and periodicities in distances between occupied antibody binding sites. AFM analysis of mucin polymers at the single molecule level provides new information about the genetic origins of individual polymers and the contributions of glycosylation to the physicochemical properties of mucins, which can be correlated with information obtained from biochemistry, antibody binding assays, and molecular biology techniques.

In this paper the surface activity of protein mucin at solution/air interface has been studied. The experiments of the adsorbed protein at solution/air interface have been carried out with a range of protein concentrations at a defined pH. The adsorption of the protein to solid surfaces and the degree of hydrophobicity at solid/solution interface of mucin have been evaluated at different pH and in the presence of Hofmeister electrolyte. The results from these studies have been further substantiated by surface potential measurements of mucin covered surface on stainless steel. Quartz crystal microbalance (QCM) has been used to follow the protein adsorption kinetics from solution to solid surface. The results from these measurements show that the adsorption behavior has a remarkable dependence on the degree of maximum coverage and is almost independent of the ionic strength. Other characteristic features such as maximum adsorption values at the protein isoelectric point (IEP4.7) and low-affinity isotherms that showed surface saturation even under unfavorable electrostatic conditions have been observed. The amount of mucin adsorbed in the presence of electrolytes has been estimated using electron spectroscopy for chemical analysis (ESCA). The study clearly shows that there exists an inverse relationship between the hydrophobicity and surface tension of the protein and also on the hydrated radius of Hofmeister electrolyte used.

Background: Tear secretion and tear film stability seem to be influenced by pseudoexfoliation syndrome (PEX). Histopathological evaluation of conjunctival goblet cells in PEX patients might explain the influence on tear film stability., Methods: In a prospective, cross-sectional study, 40 eyes of 40 patients with PEX (study group) had measurement of Schirmer test with anesthesia and break-up time prior to cataract surgery. At the end of an uneventful operation, a 1 x 1.5 mm sample of conjunctival tissue was obtained and light and electron microscopy were performed. Results were compared with a control group. Histology evaluation was masked. Primary outcome measures included Schirmer test with anesthesia, break-up time, goblet cell density and morphological character of conjunctiva and conjunctival goblet cells., Results: Both Schirmer test and break-up time values were significantly lower (P= 0.01 and P= 0.001) in the PEX patients (mean 10.6 mm and 8.6 s respectively) than in the control group (13.4 mm and 12.3 s respectively). The number of goblet cells in patients with PEX compared with the control group showed no statistically significant difference (P= 0.66). Use of trichrome stain revealed significant differences in the staining properties of goblet cells between patients with PEX and controls. On electron microscopy, typical pseudoexfoliation filaments were found in the connective tissue of conjunctiva of PEX patients. Remarkable changes of mucin pockets in the study group were also noticed; they had extremely irregular distribution, various forms of size and appearance and even complete collapse., Conclusion: Pseudoexfoliation seems to alter basic features of goblet cell morphology, thus affecting tear film stability. Further studies are necessary to determine the exact mechanism.

Purpose: The structure of mucin balls collected from silicone hydrogel contact lens wearers was examined to determine their nature., Methods: Tears containing mucin balls were collected using a capillary tube. These were processed for light microscopic histochemistry, scanning electron microscopy, and electron microscopic elemental analysis. Mucin balls were also observed in vivo using confocal microscopy., Results: Histology showed that the mucin balls were PAS positive, indicating that glycoproteins form a major component. Lipids and bacteria were not detected. Scanning electron microscopy did not show the surface to be smooth but revealed a variation in density across the surface. Elemental analysis was inconclusive., Conclusions: Mucin balls are likely to be made from collapsed mucin and are unlikely to have been formed as a result of pearling around a silicon, lipid, or bacterial kernel.

The addition of cations to the imaging buffer for AFM has been previously shown to improve the binding of biological molecules to mica. Investigations were carried out to find the concentration of NiCl(2) required to immobilize mucin molecules on a freshly cleaved mica surface, for imaging using intermittent contact in air. Drop-deposition of samples prepared in HEPES buffer with 1, 2 and 5mM NiCl(2) revealed the sensitivity of the mucin molecules to salt. Dialysis of the mucin solutions dramatically reduced the amount of salt present and allowed single molecules to be imaged, revealing a variation in thickness along their length. Spray deposition of the same mucin solutions produced single molecules that, although less affected by co-adsorbed salt, showed a degree of self-folding. This shows the sensitive balance between HEPES and NiCl(2) required for successful imaging of the sub-molecular features of individual mucin molecules.

Mucins are important glycoproteins in the mucociliary transport system of the middle ear and Eustachian tube. Little is known about mucin expression within this system under physiological and pathological conditions. This study demonstrated the expression of MUC5B, MUC5AC, MUC4, and MUC1 in the human Eustachian tube, whereas only MUC5B mucin expression was demonstrated in noninflamed middle ears. MUC5B and MUC4 mucin genes were upregulated 4.2- and 6-fold, respectively, in middle ears with chronic otitis media (COM) or mucoid otitis media (MOM). This upregulation of mucin genes was accompanied by an increase of MUC5B- and MUC4-producing cells in the middle ear mucosa. Electron microscopy of the secretions from COM and MOM showed the presence of chainlike polymeric mucin. These data indicate that the epithelium of the middle ear and Eustachian tube expresses distinct mucin profiles and that MUC5B and MUC4 mucins are highly produced and secreted in the diseased middle ear. These mucins may form thick mucous effusion in the middle ear cavity and compromise the function of the middle ear.

Intestinal neuronal dysplasia (IND) is a malformation of the enteric plexus that clinically resembles Hirschsprung's disease (HD). In HD, the aganglionic bowel is characterized by the presence of hypertrophic nerve trunks and increased numbers of adrenergic and cholinergic nerve fibers, whereas IND type B (IND-B) is characterized by dysplasia of parasympathetic nerves, hyperganglionosis, and giant ganglia. The aims of this study were to investigate the relationship between the distribution of mast cells (MC) and abnormal neuronal innervation and the impact of abnormal neuronal innervation on colonic epithelial differentiation in relation to the typical innervation abnormalities seen in HD and IND. Full-thickness rectal-biopsy or resected surgical specimens from 15 patients (7 HD, 4 IND, 4 control) were examined by conventional hematoxylin-eosin, periodic acid-Schiff Alcian blue (PAS-AB), toluidine blue staining. The aganglionic and IND-B segments had larger numbers of MC in all layers than the ganglionic and control segments. There was a close relationship between the hypertrophic nerve fibers and the distribution of the MC. In contrast to the aganglionic segments, the mucin composition of the IND-B segments was normal. This finding suggests that innervation anomalies do not reflect epithelial differentiation to the same extent.

Purpose: The goal of this study was to characterize the morphology of the mucinous layer on rabbit, bovine, owl, and human corneal endothelial cells., Methods: Corneoscleral buttons were fixed using cetylpyridinium chloride to stabilize "mucus" and the tissue was prepared for transmission electron microscopy. Photomicrographs were measured to determine the thickness of the endothelial and epithelial mucinous layer in the central cornea., Results: The endothelial mucinous layer was seen as a nearly uniform electrodense region on the apical aspect of the endothelium. It was found to be 0.9 microm, 0.9 microm, 0.9 microm, and 0.5 microm thick in rabbit, bovine, owl, and human, respectively. The owl endothelium had an additional less electrodense layer with a granular appearance and a thickness of about 200 microm. The mucinous layer on the epithelium was similar in appearance to that on the endothelium and across species., Conclusions: The morphologic similarity of the endothelial and epithelial mucinous layers is a serendipitous finding that should prove valuable in experimental design. Ultimately, it is hoped that studies of the posterior corneal surface will deepen our knowledge of endothelial protection.

In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10(7) sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to alpha-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-alpha-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.

We have isolated the high-M(r) mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average M(r) in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a 'ladder' of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. M(r) measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro.

Fractalkine (FKN), a CX(3)C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX(3)C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.

Mucin accumulation in ependymomas is thought to be limited to the myxopapillary variant and represents an important diagnostic feature. Similarly, signet-ring cells in ependymomas have been shown by electron microscopy to represent microrosette instead of mucin secretion. This study describes an infratentorial ependymoma largely composed of mucinous areas and signet-ring cells. The ependymal nature of mucin-secreting cells was confirmed by ultrastructural analysis. This case widens the variable spectrum of ependymal morphology. The value of electron microscopy in differentiating central nervous system neoplasms showing mucous secretion is stressed.

High-resolution atomic force microscopy has been applied to the imaging of intact human ocular mucins in a near-physiological buffer. The mucins displayed a range of lengths from several hundred nanometers to several microns. By varying the ionic composition of the imaging environment, it was possible to image molecules rigidly fixed to the substrate and the motion of single molecules across the substrate. From static molecular images, high-resolution line profiles show a variation of up to +/-0.75 nm in thickness along the molecule. This variation is localized in regions of several tens of nanometers. It is interpreted in terms of the varying glycosylation along the mucin and is consistent with the known size of oligosaccharides in ocular mucins. The dynamic images indicate the possibility of following mucin interactions in situ.

Saliva contains two major families of mucins (MG1 and MG2); the polypeptide of the smaller of these glycoproteins (MG2) has been assigned as the product of the MUC7 gene. In this study we have devised a rapid two-step procedure that recovers this glycoprotein essentially free of other components and in sufficient quantity to enable physical and self-interaction studies. Raw saliva was solubilized in 4 M guanidinium chloride and thereafter subjected to Sepharose CL-4B chromatography. The MG2-rich fraction was recovered free from the larger MG1 glycoproteins and also smaller proteins/glycoproteins (molecular mass less than 100 kDa). MG2 glycoproteins were finally purified by anion-exchange chromatography on Mono Q. The purity of the preparation was assessed by SDS/PAGE after radiolabelling of the molecules with [14C]acetic anhydride. Peptide mapping, N-terminal sequencing and amino acid analysis verified the polypeptide of the mucins as the MUC7 gene product. The isolated molecules were examined by electron microscopy and appeared as short flexible worm-like structures 30-120 nm in length. The distribution was heterogeneous, containing a major component with number-average and weight-average lengths of 52 and 55 nm respectively and a minor component with number-average and weight-average lengths of 94 and 98 nm respectively. We propose that the two differently sized populations represent monomeric and dimeric species of the mucins. Gel chromatography performed in 0.2 M NaCl indicated the presence of monomers, dimers and tetramers; an average molecular mass for the preparation was 192 kDa. However, in 4 M guanidinium chloride the molecular mass was 158 kDa and a similar molecular mass (155 kDa) was determined for the mucin preparation after reduction. These results suggest that the mucins might self-associate via a protein-mediated interaction. On the basis of the results a model is proposed for the self-association of the MUC7 mucin, which might be important for its biological function.

Aims: To study mucin histochemistry of the normal esophagus, esophageal adenocarcinoma, and carcinoma exhibiting glandular and squamous elements, to ascertain the origin of these tumors., Methods: Mucin histochemistry was studied in sections of the normal cardioesophageal junction obtained from 25 post-mortem specimens and in 12 mucin-secreting esophageal carcinomas., Results: The normal submucosal esophageal glands and three adenocarcinomas secreted predominantly sulfomucins; a mixture of neutral and sialomucins was seen in the nine carcinomas with squamous and glandular traits. Barrett's metaplasia was not encountered., Conclusions: In the absence of Barrett's metaplasia, esophageal adenocarcinoma probably arises from the submucosal glands, whereas squamous carcinomas with mucin-secreting component could arise from metaplastic change in squamous epithelium, cardiac glands, or multipotent stem cells in the epithelium.

We present a case of (arterio) venous hemangioma of 15 years' duration, which arose in a 43-year-old Japanese man on his right infraorbital area. Elastic stain and electron microscopy did not show any evidence for arterial components. Some thick-walled vessels showed mucin deposition and wide intercellular spaces between smooth muscle cells. Several glomoid cells were encountered among these cells. Although the histogenesis of (arterio) venous hemangioma is controversial, the presence of glomoid cells and the mucin deposition, which is occasionally detected in glomus tumors, suggest the relation of this tumor to the Suquet-Hoyer canal.

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' (omega), and the dynamic complex viscosity, eta*(omega), are observed for lipid-containing CSM. This 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.

A case of metachronous cancer of gastrointestinal tract is reported. Three poorly differentiated malignant epithelial tumours developed in a 27-year-old patient within three years. They located in the stomach, small and large intestine. Light-microscopically, the tumours were formed mainly of non-differentiated rounded cells with occasional signet ring cells. At electron microscopic examination the presence of mucin granules was confirmed. Moreover, a variable amount of electron-dense endocrine-like granules was found in tumour cells. According to some publications, endocrine differentiation of gastrointestinal cancers is considered to be a poor prognostic feature, hence electron microscopy or special staining when possible can be important in the evaluation of prognosis.

Airway mucus from asthmatics is often unusually solid. The death of a patient in status asthmaticus allowed the collection of 28 g of abnormal airway mucus at autopsy. Its chemical and physical properties were studied to reveal differences from more normal airway mucus. The gel plug taken from the airways could be dispersed in 6 M guanidinium chloride, but it took > 1 wk and 700 ml of extractant to disperse 3 g of exudate completely. In contrast, treatment with 10 mM dithiothreitol, which reduces disulfide bonds, dispersed the gel within seconds. Mucins accounted for 25% of the non-dialyzable material in the gel, while DNA constituted < 1% and proteoglycans could not be detected. The mucins were similar in architecture and general composition to other respiratory mucins and were present at a high concentration (approximately 40 mg/ml). The majority of mucins were of extreme size (mean M(r) 30-40 x 10(6)) and slow to dissolve, but sequential extraction experiments on the gel exudate demonstrated a proportion of mucins (15%), the most readily extracted, which had a higher density, 1.45-1.55 g/ml, a lower M(r) (11.5 x 10(6)) and were markedly more acidic than the bulk of the mucins. Both major and minor mucin populations were extremely heterogeneous in mass distribution. Electron microscopy of the major mucin species demonstrated extensive networks of molecules many microns in length. The major mucin species was distinctly less acidic than mucins previously described from either normal or diseased airways. Amino acid analysis of fractions across the charge distribution suggested the presence of at least two different mucin proteins occurring as distinct glycoforms.

alpha-Dystroglycan has been isolated from chicken cardiac muscle and its molecular weight was estimated to be approximately 135 kDa. The avian protein interacts with murine Engelbreth-Holm-Swarm (EHS) tumor laminin via interaction with the C-terminal LG4 and LG5 domains (fragment E3) of the laminin alpha-chain. This laminin binding is calcium-dependent and can be competed by heparin. Electron microscopy investigation on the shape of alpha-dystroglycan suggests that the core protein consists of two roughly globular domains connected by a segment which most likely corresponds to a mucin-like central region also predicted by sequence analysis on mammalian isoforms. This segment may act as a spacer in the dystrophin-associated glycoproteins complex exposing the N-terminal domain of alpha-dystroglycan to laminin in the extracellular space.

Calcium hydroxyapatite (HAP) crystals formed in vitro in the presence of polymeric human gallbladder mucin (1.0 mg/mL) were smaller (0.75 +/- 0.39 microns) than control crystals (7.86 +/- 2.76 microns), but the mucin did not affect the kinetics of crystal formation or alter the amount of mineral phase present at equilibrium. In contrast, glycopeptide subunits produced by proteolysis of the native mucin had no effect on HAP crystal size. Both native mucin and glycopeptides bound to mature HAP crystals, but the glycopeptides were much more readily displaced by phosphate ions. Therefore, in experiments where HAP was being formed, the phosphate ions inhibited the interaction of glycopeptides with the nascent HAP. These results indicate that gallbladder mucin may modulate HAP formation in vivo, and that this ability may be altered during pathological states, such as neutrophil infiltration or bacterial colonization, that may cause the release of proteinases capable of digesting mucin.

A polychrome method for detection of mucin substance in paraffin section is produced by sequential stepwise staining of hematoxylin, crude lac extract (Laccifer lacca), and crude curcuma extract (khamin shan-Curcuma longa). The name LacCur stain is proposed. After a tissue section is deparaffinized and rehydrated, it is stained with Weigert's hematoxylin for 7 minutes. After a quick wash, it is stained for at least 3 hours with lac dye mordanted with aluminum chloride. Washed again and premordanted with ferric chloride for 1 minute, in the last step, it is counterstained with curcuma dye for 5 minutes. With this staining method, the nuclei are stained black, mucin deep red, and organelles and ground substances brownish yellow. The method and outcome colors are comparable to the widely used Mayer's mucicarmine staining method. It costs less than the Mayer's mucicarmine staining method and the procedure is not complicated.

Two populations of reduced subunits were present in the mucins purified from pooled normal secretions and asthmatic and chronic bronchitic sputa; their relative level differed between samples. To investigate the nature of this heterogeneity, an asthmatic respiratory mucin preparation from a single individual was reduced and alkylated with 14C-iodoacetamide. This preparation was analyzed by gel filtration, agarose gel electrophoresis, immunoblotting, rate-zonal- and density-gradient centrifugation, and HPLC ion-exchange- and reverse-phase chromatography. Two populations (A and B) of reduced mucin subunits and a high-M(r)protein-rich fraction were identified. Species A has the higher molecular mass, is slowest migrating on agarose electrophoresis, has longer oligosaccharide chains, and expresses the carbohydrate structure sialyl-Le(x). Species B has a lower molecular mass, migrates faster in agarose electrophoresis Species B has a lower molecular mass, migrates faster in agarose electrophoresis, has shorter chains, and does not express sialyl-Le(x). The two subunits have similar but not identical amino acid compositions and 14C-tryptic peptide maps indicating they have different protein cores. The anti-sialyl-Le(x) antibody selectively precipitated subunit A not only from the reduced but also from the nonreduced mucin preparation, demonstrating that subunits A and B are present in different intact mucins.

The prostate gland normally secretes neutral mucosubstances that can be detected within the lumina of acini and ducts; adenocarcinomas often produce both acidic and neutral mucins, a feature that has been suggested to be of some diagnostic use. The presence of mucin-filled cells is not, however, a feature of the normal prostate. Over the last few years, we have observed tall, columnar, mucin-secreting cells in a variety of conditions in 12 benign prostates. All cases were stained histochemically for mucin with Mayers' mucicarmine, alcian blue (pH 2.7), and periodic-acid-Schiff with diastase digestion. In four cases, immunoperoxidase stains for prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) were performed. Mucin-secreting cells were found in the foci of sclerotic atrophy (n = 5), transitional cell metaplasia (n = 3), basal cell hyperplasia (n = 2), prostatrophic hyperplasia (n = 1), and nodular hyperplasia (n = 1). In all examples, the cells stained intensely with PAS, mucicarmine, and alcian blue. The cells were nonreactive for PSA and PAP in the cases studied. To our knowledge, the presence of tall, columnar, mucin-secreting cells has not been previously described in atrophy or basal cell hyperplasia. These observations expand our appreciation of the histologies that may be seen in the prostate gland; in addition, the recognition of acidic mucin-secreting cells in benign lesions points to the nonspecificity of this finding in the diagnosis of malignancy.

Human polymorphic epithelial mucin is a high-molecular-mass glycoprotein that associates to provide protection to the epithelial-cell surface and may afford the malignant cell a selective advantage for growth. The scanning-tunnelling-microscopy micrographs obtained in the present study identify the purified human ovarian-carcinoma polymorphic epithelial mucin glycoproteins as rod-shaped molecules of mixed length. The dimensions of the individual molecules range from 25 to 45 nm in length and are 3-4 nm in width. The images further suggest that lateral association of the rods occurs.

Purified germ-free rat intestinal mucin was found by chemical analysis to contain 25% protein, enriched in serine, threonine, and proline, 75% carbohydrate, and no nucleic acid. It was analyzed by darkfield electron microscopy and found to consist of long filamentous molecules with a maximum length of approximately 740 nm, a mean length of 456 nm, and a mean width of 7 nm. Given reasonable assumptions derived from earlier work on other well-characterized mucins, the molecular weight of the peptide, calculated by the length from electron microscopy, was 200,000, and, given the chemical composition, the molecular weight of the entire mucin molecule was calculated to be approximately 800,000.

Oligomeric gastric mucin was isolated from the fundic part of the rat stomach. Previously we have shown by biochemical analysis that this oligomeric mucin consists of disulphide-linked homo-oligomers, which contain no other covalently attached proteins [Dekker, Aelmans & Strous (1991) Biochem. J. 277, 423-427]. Electron-microscopic images of the oligomeric mucin revealed a heterogenous population of long filamentous molecules of 300-3000 nm length. After reduction and carboxymethylation the monomeric mucins displayed a length distribution with a single peak at about 279 nm. Length-distribution analysis of oligomeric molecules with length up to 1000 nm revealed three subpopulations with one, two or three times the length of the monomeric mucin. The oligomers displayed small globular domains of about 15 nm, which were equally spaced along the molecule's length. As the distance between these globular domains was similar to the monomer length, these domains most likely indicate attachment sites of the monomers. These results show that the mucin monomers attached end-to-end in the oligomer. Biosynthesis of the mucin oligomers was studied by labelling of stomach explants in vitro with [35S]methionine, [3H]galactose or [35S]sulphate and subsequent immunoprecipitation of the mucin with a specific antiserum. Analysis by electrophoresis and gel filtration revealed that the oligomerization takes place by formation of disulphide bonds between the 300 kDa mucin precursors. The mucin was exclusively synthesized and secreted as fully glycosylated oligomers, as neither precursor proteins nor monomeric mucin were detected in the culture medium. A model for the biosynthesis of rat gastric mucin is proposed in which the filamentous mucin monomers are linked end-to-end by disulphide bonds.

Mucus glycoproteins (mucins) were isolated from sputum of patients with cystic fibrosis (CF) after separation into sol and gel phases. The mucus gel was solubilized with gentle stirring in 6 M-guanidinium chloride supplemented with proteinase inhibitors, and purification of mucins was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. Density-gradient centrifugation also revealed a heterogeneity of the macromolecules, the pattern of which varied between individuals, and mucins from the gel phase was pooled as 'heavy' and 'light' fractions. Gel chromatography on Sepharose CL-2B showed that the heavy fraction contained a larger proportion of smaller species than the 'light' fraction and that the gel phase mucins were much larger than those from the sol. An apparently homogeneous high-Mr mucin population from one individual contained approx. 70% (w/w) carbohydrate, the major sugars being N-acetylglucosamine (17.8%), N-acetylgalactosamine (6.7%), galactose (20.7%), fucose (13.2%) and sialic acid (11.4%). These mucins had an S020.w of 47 S, and an Mr of 15 x 10(6) -20 x 10(6), and rate-zonal centrifugation revealed a polydisperse size distribution [range (5-30) x 10(6)] with a weight-average Mr of 17 x 10(6). The whole mucins were visualized with electron microscopy as linear and apparently flexible threads, disperse in size. Reduction produced subunits which were included on Sepharose CL-2B, and subsequent trypsin digestion yielded high-Mr glycopeptides which were further retarded. The size distributions and fragmentation patterns of mucin from two other CF patients were the same, as studied by gel chromatography, rate-zonal centrifugation and electron microscopy. We conclude that CF mucins are heterogeneous in both size and buoyant density and that the various populations, though differing in buoyant density, share the same architecture and macromolecular properties and are, in this respect, similar to mucins from normal respiratory secretions [Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt (1990) Biochem. J. 265, 179-186] and human cervical mucus [Carlstedt & Sheehan (1989) SEB Symp. XLIII 289-316].

Polyclonal antibodies were raised in rabbits towards reduced subunits of human cervical mucus glycoproteins. The reduced subunits almost completely inhibited the antiserum, whereas the intact mucins and the heavily glycosylated fragments obtained after digestion of reduced subunits with trypsin (T-domains) caused only partial inhibition. Periodate oxidation of intact mucins, reduced subunits and T-domains caused no effect on the antibody response, and fragments obtained by more extensive proteolysis of the reduced subunits (P-domains) showed no inhibitory activity. By using electron microscopy, antibodies from T-domain-adsorbed antisera were revealed as bound to cervical mucin reduced subunits, either directly or with colloidal gold-Protein A. Binding sites (100-150 nm apart) were observed at the ends and at internal positions of the reduced subunits. We conclude that the antibodies do not recognize carbohydrate structures but are directed to two kinds of protein epitopes, one shared by whole mucins, reduced subunits and T-domains, and the other specific to the reduced subunit fragment. The latter epitopes are 'cryptic' and are probably shielded within folded protein domains stabilized by disulphide bonds. Human bronchial, cervical, gastric and salivary mucus glycoproteins share some of these cryptic epitopes.

Mucine-like glycoproteins have been studied by structural indication. Textures of these compounds have been described. Possibility for using mucines as matrix structures in microcrystallographic method of analysis of complex multicomponent systems has been discussed.

A case of pulmonary carcinoma with dual (glandular and endocrine) differentiation diagnosed by fine needle aspiration cytology is reported. By light microscopy, the neoplastic cells showed mucicarmine, periodic-acid Schiff, and Grimelius silver positivity. On immunostaining, neuron specific enolase, chromogranin, calcitonin and serotonin were demonstrated. Electron microscopy revealed two different types of distinctive cytoplasmic inclusions in the same cell, namely: carcinoid type of neurosecretory granules and mucin droplets. Diagnostic criteria and possible histogenesis of this rare tumour entity are discussed.

Mucins represent the main components of gel-like secretions, or mucus, secreted by mucosae or some exocrine glands. These high-molecular-weight glycoproteins are characterized by the large number of carbohydrate chains O-glycosidically linked to the peptide. The determination of mucin molecular weight and conformation has been controversial for several reasons: 1) the methods used to solubilize mucus and to purify mucins are different and 2) the molecules have a strong tendency to aggregate or to bind to other molecules (peptides or lipids). Recently, electron microscopy has shown the filamentous shape of most mucins and their polydisperse character which, in some secretions, might correspond to a polymorphism of the peptide part of these molecules. The recent development of high pressure liquid chromatography and high-resolution proton NMR spectroscopy has allowed major progress in the structural study of mucin carbohydrate chains. These chains may have from 1 to about 20 sugars and bear different antigenic determinants, such as A, B, H, I, i, X, Y or Cad antigens. In some mucins, such as human respiratory mucins, the carbohydrate chain diversity is remarkable, which raises many questions. Mucins are molecules located at the interface between mucosae and the external environment. The carbohydrate chain diversity might allow many interactions between mucins and microorganisms and play a major role in the colonization or the defense of mucosae.