Skin mucus proteins of lumpsucker (Cyclopterus lumpus)

Fish skin mucus serves as a first line of defense against pathogens and external stressors. In this study the proteomic profile of lumpsucker skin mucus was characterized using 2D gels coupled with tandem mass spectrometry. Mucosal proteins were identified by homology searches across the databases SwissProt, NCBInr and vertebrate EST. The identified proteins were clustered into ten groups based on their gene ontology biological process in PANTHER (www.patherdb.org). Calmodulin, cystatin-B, histone H2B, peroxiredoxin1, apolipoprotein A1, natterin-2, 14-3-3 protein, alfa enolase, pentraxin, warm temperature acclimation 65 kDa (WAP65kDa) and heat shock proteins were identified. Several of the proteins are known to be involved in immune and/or stress responses. Proteomic profile established in this study could be a benchmark for differential proteomics studies.     

Human duodenal gland (Brunner's gland) mucus glycoprotein analysis

A mucus glycoprotein of the duodenal gland is characterized. The glycoprotein was isolated from a water-soluble homogenate fraction of the submucosal tissue of the most proximal part of the small intestine, containing the duodenal gland, and was purified from contaminating protein by two sequential equilibrium-centrifugation steps in CsCl density gradients. Structural analysis of the purified glycoprotein showed two regions in the protein core: one part characterized by the presence of essentially all of the cysteine residues and another by the presence of most of the serine and threonine. Carbohydrate was found linked to the latter part. Rat (H. L. Smits, P. J. M. van Kerkhof, and M. F. Kramer (1982) Biochem. J. 203, 779-785.) and human duodenal gland mucus glycoprotein show homology in chemical composition. Both glycoproteins have a relatively high protein content and contain little sulfate and no neuraminic acid. In man the mucus glycoprotein, however, has a higher content of serine plus threonine, a lower content of N-acetylglucosamine, a slightly higher content of fucose, and a lower molar ratio of N-acetylgalactosamine relative to serine plus threonine.     

Horned lizards (Phrynosoma) incapacitate dangerous ant prey with mucus

Horned lizards (Iguanidae, Phrynosomatinae, Phrynosoma) are morphologically specialized reptiles characterized by squat, tank-like bodies, short limbs, blunt snouts, spines and cranial horns, among other traits. They are unusual among lizards in the degree to which they specialize on a diet of ants, but exceptional in the number of pugnacious, highly venomous, stinging ants they consume, especially harvester ants (genus Pogonomyrmex). Like other iguanian lizards, they capture insect prey on the tongue, but unlike other lizards, they neither bite nor chew dangerous prey before swallowing. Instead, they employ a unique kinematic pattern in which prey capture, transport and swallowing are combined into a single feeding stage, apparently leaving the mouth, pharynx, esophagus and stomach vulnerable to bites and stings. Nevertheless, horned lizards consume dozens of harvester ants without harm. We show that their derived feeding kinematics are associated with unique, mucus-secreting pharyngeal papillae that apparently serve to immobilize and incapacitate dangerous ants as they are swallowed by compacting them and binding them in mucus strands. Radially branched esophageal folds provide additional mucus-secreting surfaces the ants pass through as they are swallowed. Ants extracted from fresh-killed horned lizard stomachs are curled ventrally into balls and bound in mucus. We conclude that the pharyngeal papillae, in association with a unique form of hyolingual prey transport and swallowing, are horned lizard adaptations related to a diet of dangerous prey. Harvester ant defensive weapons, along with horned lizard adaptations against such weapons, suggest a long-term, predator-prey, co-evolutionary arms race between Phrynosoma and Pogonomyrmex. J. Exp. Zool. 309A:447-459, 2008. (c) 2008 Wiley-Liss, Inc.     

Isolation and partial characterization of rat duodenal-gland (Brunner''s-gland) mucus glycoprotein.

A mucus glycoprotein was isolated from the duodenal glands of the rat and purified by repeated density-gradient centrifugation. The characterized glycoprotein is unique to the mucous cells of the duodenal glands and is not present in parts of the small intestine devoid of these glands. The chemical composition of the purified glycoprotein is characteristic for glycoproteins of the mucin-type. Its protein content is relatively high and amount to 35% by weight. No neuraminic acid and little sulphate (2%) is present. Evidence is presented that the native glycoprotein is built up from subunits held together via disulphide bridges in a non-glycosylated region of the protein core.     

Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss)

AIMS: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. METHODS AND RESULTS: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. CONCLUSIONS: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.     

Drag-reducing properties of bacteria from the skin mucus of the cornetfish (Fistularia commersonii)

Scanning electron microscopy and fluorescence microscopy after scanning with DAPI indicated that the skin mucus of the cornetfish contained large numbers of bacteria, 4 × 10⁸ per cm3. However, viable counts yielded only 2 × 104 per cm³. Twelve bacterial strains were isolated directly from the mucus and another ten strains were obtained following enrichment on pasteurized mucus medium. Most of the isolates belonged to the genus Pseudomonas; a smaller number were classified as Micrococcaceae. Cultures of 13 of the isolates were active in reducing friction in a turbulent flow rheometer. The surface active and drag-reducing properties of three strains—JR5, JR8, and GB7—were studied further. The drag-reducing activities, which were extracellular, were concentrated by ultra-filtration. The chemical composition of the concentrated preparations consisted of 14–24% protein and 38–75% polysaccharide. The major components of the polysaccharide fraction were galacturonic acid, galactosamine, and glucosamine, with lesser amounts of glucose and galactose. The most active preparation, from strain JR8, had a specific drag-reducing activity of 77 units per mg. Strain JR5 was the most hydrophobic as measured by the DOS and BATH tests. JR8 gave intermediate values, and GB8 showed low hydrophobicity values in both tests. The hydrocarbon-in-water emulsifying ability of the concentrated polymer fractions from JR8, GB7, and JR5 were high, intermediate, and low, respectively. The emulsifying and drag-reducing activities of the polymer fraction from strain JR8 were separated from each other by extraction with hydrocarbons. The emulsifying activity was due to a carbohydrate-protein complex, whereas the drag-reducing activity was associated with a uronic acid-containing polysaccharide. Offprint requests to: E. Rosenberg.     

Carbohydrate-binding site of a novel mannose-specific lectin from fugu (Takifugu rubripes) skin mucus

Pufflectin-s, identified in the skin mucus of the fugu Takifugu rubripes, is a novel mannose-specific lectin with similar structure to monocotyledonous plant lectins. In the present study, mutational analysis was used to reveal the mannose-binding sites of pufflectin-s. Putative binding sites were mutated as follows: binding site 1; rPL-D32E (Asp³² → Glu³²), rPL-N34S (Asn³⁴ → Ser³⁴) and rPL-V36A (Val³⁶ → Ala³⁶) whereas binding site 2; rPL-D61E (Asp⁶¹ → Glu⁶¹), rPL-N63S (Asn⁶³ → Ser⁶³) and rPL-V65A (Val⁶⁵ → Ala⁶⁵). All recombinant proteins were expressed in Escherichia coli, purified with two chromatographic steps, and then subjected to mannose-binding assay by affinity chromatography. Recombinant wild-type pufflectin-s (rPL-wt) as well as three mutants with changes in binding site 2 could bind to mannose, in contrast to the three mutants with changes in binding site 1 in which mannose-binding activity was completely lost. These results clearly demonstrate that, at the least, binding site 1 is critical to mannose-binding activity in pufflectin-s.     

Changes of 31P metabolism during mucus secretion in the slug (Incilaria bilineata)

1. Changes in the amounts of mucus secreted by the epidermis of the slug in response to an irritant chemical substance (NaCl) and the effect of mechanical stimulation on high-energy phosphorylation metabolism were studied.2. ³¹P signals obtained from the slug with in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy included phosphonate, sugar phosphate (SP), inorganic phosphate (Pi), arginine phosphate (Arg-P), and ATP (γ, α and β).3. When the slug epidermis came into contact with NaCl or was exposed to mechanical stimulation, the concentration of Arg-P decreased and that of Pi increased along with mucus secretion.4. The change of ³¹P signals in response to chemical stimulation was much more variable than that in response to mechanical stimulation.5. The β-ATP concentration was dependent on the amount of mucus secreted.     

The Universal Plausibility Metric (UPM) & Principle (UPP)

Background

Mere possibility is not an adequate basis for asserting scientific plausibility. A precisely defined universal bound is needed beyond which the assertion of plausibility, particularly in life-origin models, can be considered operationally falsified. But can something so seemingly relative and subjective as plausibility ever be quantified? Amazingly, the answer is, "Yes." A method of objectively measuring the plausibility of any chance hypothesis (The Universal Plausibility Metric [UPM]) is presented. A numerical inequality is also provided whereby any chance hypothesis can be definitively falsified when its UPM metric of ξ is < 1 (The Universal Plausibility Principle [UPP]). Both UPM and UPP pre-exist and are independent of any experimental design and data set.

Conclusion

No low-probability hypothetical plausibility assertion should survive peer-review without subjection to the UPP inequality standard of formal falsification (ξ < 1).     

Evaluation of non-specific immune components from the skin mucus of olive flounder (Paralichthys olivaceus)

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish under intensive aquaculture and at prolonged low temperatures from relevant infections due to inadequate adaptive immune responses. In the present study we aimed to understand the mucosal immunity of an economically important mariculture fish, olive flounder (Paralichthys olivaceus) by evaluating the immune components from its skin mucus. The activities of lysozyme (233.33+/-171.82 units mg(-1)), trypsin-like protease (42.84+/-1.249 units mg(-1)), alkaline phosphatase (0.376+/-0.005 units mg(-1)) and esterase (0.170+/-0.006 units mg(-1)) were detected in the skin mucus. Transferrin was identified by MALDI-TOF/MS analysis. ELISA and immunoblot assays using anti-flounder IgM monoclonal antibody showed the presence of a significant level (1.80+/-0.001, n=3) of monomer immunoglobulin M (IgM) with approximate molecular weight of 160 and 25 kDa under non-denaturing and denaturing states, respectively. Skin mucus showed strong antibacterial activity against tested fish pathogenic bacteria. In addition, skin mucus successfully agglutinated (HA titre 2(8)), but completely failed to haemolyse, rabbit erythrocytes. In conclusion, the major immune components of the skin mucus, identified in the present study, are possibly involved in the broad spectrum non-specific immunity of olive flounder.     

Structural forms and possible roles of aligned cytoskeletal biopolymers in hagfish (slime eel) mucus

The hagfish, marine benthic dwellers, are primitive vertebrates which, upon stress, secrete massive quantities of mucus from slime glands of the integument by the holocrine mode. Lysis of each gland thread cell releases a single, intermediate filament-rich thread ( 60 cm × 1.5 μm), which collectively modulates the hydration and cohesiveness of mucus. In this report, with the Pacific hagfish (Eptatretus stouti), we showed that the threads exhibit a tendency for self-assembly into novel, aligned and intertwined cytoskeletal aggregates (“cables”). Large cables (15–40 cm in length, 50–800 μm in width) can be physically prepared from the whole mucous exudate, but similar smaller cables (0.1–10 cm in length, 50–200 μm in width) were found occurring naturally, particularly at the ends of laid eggs. Threads and cables may facilitate adhesion of mucus to the integument of the fish, restrict eggs to an optimum spawning site, and abet defense against invasive organisms.     

Preserving the viscous coral surface mucus layer using low-acid glycol methacrylate (GMA) resin

The surface mucus layer (SML) is of critical importance in health, disease, and stress responses of corals; however, visualising the intact SML is challenging. Dehydration during histological preparation causes shrinkage and deformation of the mucus gel layer, while fragile, unattached mucus exudates are typically lost altogether. Here, we describe a novel technique using water-soluble glycol methacrylate resin embedding that more accurately preserves the in situ SML. Thickness of the preserved SML is similar to that previously measured on live corals using a glass microprobe. The technique allows microscopic visualisation of the SML structure, as well as thickness and continuity measurements, which are important indicators of SML function in health and disease.

     

Keratin-like components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stouti)

Summary The hagfishes (cyclostomes) are known to secrete copious amounts of mucus mainly by the holocrine mode from the slime glands. Stressed animals release two types of cells (gland thread cells, GTCs; gland mucous cells, GMCs) which rupture on contact with water and rapidly form a mass of viscous mucus. Herein we report some key sequential events of this process and document a novel role for cytoskeletal polymers. After electrostimulation of Pacific hagfish (Eptatretus stouti), the exudate was collected in a stabilization buffer and GTCs segregated from GMC vesicles. Water was added progressively to mixtures of known quantities of these entities. The changing mucous composition and properties were monitored by light- and electron microscopy, viscometry and immunogold assay. Sequentially, the threads uncoil from GTCs, aggregate with the vesicles, the vesicles rupture and release mucin-like substances, at least some of which adhere to the thread. It was found that the intermediate filament (IF)-rich threads markedly facilitate hydration and modulate the viscoelastic and cohesive properties of the resultant mucus. It was speculated that the thread abets localization of mucus in an aqueous environment and promotes adhesion of mucus to surfaces such as the fish integument. As judged by immunostaining in situ, GTCs, as well as several cell-types in the epidermis, contain keratin-like components. The role of biopolymers on the properties of teleost and mammalian mucus is discussed.