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

1988 ◽  
Vol 66 (5) ◽  
pp. 367-373 ◽  
Author(s):  
Sum P. Lee ◽  
Jane F. Nicholls ◽  
Anthony M. Roberton ◽  
Han Z. Park
Keyword(s):  
Periodic Acid ◽  
Gastric Mucin ◽  
Low Density ◽  
Gastric Mucus ◽  
Experimental Conditions ◽  
Periodic Acid Schiff ◽  
Pepsin Digestion ◽  
Cscl Density Gradient ◽  
Peptic Digestion

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

scholarly journals SPECTROPHOTOMETRY OF THE PERIODIC ACID-SCHIFF REACTION WITH PITUITARY HORMONES IN VITRO AND IN HISTOLOGICAL SECTIONS

1962 ◽  
Vol 13 (2) ◽  
pp. 239-247 ◽  
Author(s):  
Sally B. Fand ◽  
Bo Thorell
Keyword(s):  
Periodic Acid ◽  
Periodate Oxidation ◽  
Quantitative Measure ◽  
Pituitary Hormones ◽  
Experimental Conditions ◽  
Human Pituitary ◽  
Periodic Acid Schiff ◽  
Histological Sections ◽  
Schiff Reaction

The spectral light absorption of the in vitro periodic acid-Schiff reactions of 4 purified pituitary hormones is described. The absorption spectra present a maximum between 560 and 565 mµ. The color developed conforms with Beer's law for the ranges of concentration examined. The different hormones exhibit different chromogenicity per unit of biological activity: the color produced by 1 unit of FSH is equivalent to approximately 2 of TSH, 4 of LH, and 30 of ACTH. Microspectrophotometric measurements of the PAS-positive structures in histological sections of the human pituitary give absorption curves with shapes similar to those obtained in vitro, although quantitative differences exist. It is concluded that under the proper experimental conditions microspectra of the pituitary structures might, in the future, prove to give a quantitative measure of aldehyde groups generated from glycoprotein tropins by periodate oxidation.

Low doses of ethanol have Ca2+ ionophore-like effects on apical membrane potential of in vitro Necturus antrum

1991 ◽  
Vol 261 (1) ◽  
pp. G92-G103
Author(s):  
M. J. Rutten ◽  
C. D. Moore
Keyword(s):  
Membrane Potential ◽  
Apical Membrane ◽  
Periodic Acid ◽  
Ethanol Exposure ◽  
Membrane Potentials ◽  
Antral Mucosa ◽  
Low Doses ◽  
Periodic Acid Schiff ◽  
Induced Changes

The effects of low doses of luminal ethanol on the amiloride-sensitive apical membrane potential of Necturus antral mucosa were studied using conventional microelectrode techniques. Luminal ethanol (0.250-4.0% vol/vol) caused a dose-dependent hyperpolarization of the apical membrane potential (Vmc), an increase in transepithelial resistance (Rt) and resistance ratio (Ra/Rb), and a decrease in transepithelial potential (Vms). Luminal amiloride (100 microM) to 4% ethanol-treated antra did not cause any additional hyperpolarization of Vmc. Compared with luminal 2% ethanol-Ringer, an equivalent osmotic mannitol solution depolarized Vmc and basolateral potential (Vcs), decreased Rt and Ra/Rb, and increased Vms. A single dose of 0.50% ethanol attenuated the effects of a second 2% ethanol exposure on Vmc. No change in periodic acid-Schiff (PAS)-positive mucous granule content could be found between control and 2% ethanol-treated antra. The Ca2+ ionophores A23187 or ionomycin (0.25-5.0 microM) dose dependently hyperpolarized the Vmc and Vcs, increased Rt and Ra/Rb, and decreased Vms. Luminal Ca(2+)-free Ringer had no effect on luminal 2.00% ethanol-induced changes in membrane potentials or resistances. Pretreatment with BAPTA blocked by approximately 70 and 55% the Vmc hyperpolarization of 2 and 4% ethanol, respectively. Pretreatment with ruthenium red (10-50 microM) also dose dependently reduced the 2% ethanol-induced changes in Vmc. The data indicate that 1) low doses of luminal ethanol and Ca2+ ionophores have similar effects on Necturus gastric antral membrane potentials and resistances, 2) ethanol-induced hyperpolarizations of the Vmc are partially mediated through an alteration in intracellular Ca2+, and 3) low doses of luminal ethanol do not cause the release of antral epithelial mucous granules at the time when significant changes are occurring in the Vmc.

A novel method for the visualization of the in situ mucus layer in rat and man

1998 ◽  
Vol 95 (1) ◽  
pp. 97-106 ◽  
Author(s):  
NICOLA JORDAN ◽  
JULIA NEWTON ◽  
JEFFREY PEARSON ◽  
ADRIAN ALLEN
Keyword(s):  
Gastric Mucosa ◽  
Alcian Blue ◽  
Periodic Acid ◽  
Blue Staining ◽  
Mucus Layer ◽  
Gastric Mucus ◽  
Periodic Acid Schiff ◽  
Novel Method ◽  
The Mean ◽  
Thickness Measurements

1.The observed thickness of the gastric mucus barrier varies widely, even appearing discontinuous, depending on the methods used. Here we describe the development and application of a modified periodic acid Schiff/Alcian Blue staining technique for use on cryostat sections of gastric mucosa. This technique for the first time enables the preservation and visualization of the full thickness of the adherent gastric mucus layer and the underlying mucosa. 2.In designing this novel method we have selected those procedures which would result in the least alteration to the mucus layer. The methods used were snap freezing, cryostat sectioning of the whole stomach followed by brief ethanol pretreatment (10 min in 100% ethanol), a prolonged staining with periodic acid Schiff/Alcian Blue (15 min and 2.5 h respectively), a gentle post-fixation (45 min paraformaldehyde vapour at 37 °C) and the use of a water-soluble mountant. 3.A continuous, adherent mucus layer was observed over the surface of the rat gastric mucosa (periodic acid Schiff/Alcian Blue stained) and human gastric antral biopsies (periodic acid Schiff stained). In the rat the mean (S.D.) mucus thickness measurements along the antrum to oesophageal axis (which was divided histologically into four regions, A to D) were: A, 166 (47) μm; B, 179 (48) μm; C, 184 (50) μm; D (the non-glandular stratified epithelium at the top of the stomach), Absent. In human gastric antral biopsies the mean (S.D.) mucus thickness was 144 (52) μm. 4.This new technique has enabled the visualization and precise measurement of thickness of the gastric mucus layer in rat and man. The adherent gastric mucus layer was observed to be continuous in the rat glandular stomach and human antrum. In validation experiments in rat the mean mucus thickness measurements were found to be twice those measured by conventional histological techniques, in which the mucus layer appeared discontinuous and patchy. However, they were within the range of thickness values seen in unfixed tissues and in the rat in vivo preparation.

scholarly journals Necroptosis Contributes to Urban Particulate Matter-Induced Airway Epithelial Injury

2018 ◽  
Vol 46 (2) ◽  
pp. 699-712 ◽  
Author(s):  
Feng Xu ◽  
Man Luo ◽  
Lulu He ◽  
Yuan Cao ◽  
Wen Li ◽  
...  
Keyword(s):  
Particulate Matter ◽  
Pulmonary Inflammation ◽  
Periodic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Periodic Acid Schiff ◽  
Mucus Production ◽  
Gm Csf ◽  
Mucin Expression

Background/Aims: Necroptosis, a form of programmed necrosis, is involved in the pathologic process of several kinds of pulmonary diseases. However, the role of necroptosis in particulate matter (PM)–induced pulmonary injury remains unclear. The objective of this study is to investigate the involvement of necroptosis in the pathogenesis of PM-induced toxic effects in pulmonary inflammation and mucus hyperproduction, both in vitro and in vivo. Methods: PM was administered into human bronchial epithelial (HBE) cells or mouse airways, and the inflammatory response and mucus production were assessed. The mRNA expressions of IL6, IL8 and MUC5AC in HBE cells and Cxcl1, Cxcl2, and Gm-csf in the lung tissues were detected by quantitative real-time RT-PCR. The secreted protein levels of IL6 and IL8 in culture supernatants and Cxcl1, Cxcl2, and Gm-csf in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA). We used Western blot to measure the protein expressions of necroptosis-related proteins (RIPK1, RIPK3, and Phospho-MLKL), NF-κB (P65 and PP65), AP-1 (P-c-Jun and P-c-Fos) and MUC5AC. Cell necrosis and mitochondrial ROS were detected using flow cytometry. In addition, pathological changes and scoring of lung tissue samples were monitored using hemoxylin and eosin (H&E), periodic acid-schiff (PAS) and immunohistochemistry staining. Results: Our study showed that PM exposure induced RIP and MLKL-dependent necroptosis in HBE cells and in mouse lungs. Managing the necroptosis inhibitor Necrostatin-1 (Nec-1) and GSK’872, specific molecule inhibitors of necroptosis, markedly reduced PM-induced inflammatory cytokines, e.g., IL6 and IL8, and MUC5AC in HBE cells. Similarly, administering Nec-1 significantly reduced airway inflammation and mucus hyperproduction in PM-exposed mice. Mechanistically, we found PM–induced necroptosis was mediated by mitochondrial reactive oxygen species-dependent early growth response gene 1, which ultimately promoted inflammation and mucin expression through nuclear factor κB and activator protein-1 pathways, respectively. Conclusions: Our results demonstrate that necroptosis is involved in the pathogenesis of PM–induced pulmonary inflammation and mucus hyperproduction, and suggests that it may be a novel target for treatment of airway disorders or disease exacerbations with airborne particulate pollution.

Stimulation of secretion into human and feline airways by Pseudomonas aeruginosa proteases

1991 ◽  
Vol 70 (5) ◽  
pp. 2259-2267 ◽  
Author(s):  
M. Somerville ◽  
P. S. Richardson ◽  
A. Rutman ◽  
R. Wilson ◽  
P. J. Cole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Protease ◽  
Periodic Acid ◽  
Surface Epithelium ◽  
Dependent Manner ◽  
Periodic Acid Schiff ◽  
Concentration Dependent Manner ◽  
Human Bronchus ◽  
Secretion Inhibition

We have investigated the effect of elastase and alkaline protease from Pseudomonas aeruginosa on airway secretion into the trachea of anesthetized cats and from human bronchial mucosa in vitro. Secretory macromolecules were radiolabeled biosynthetically with two precursors in the cat, [3H]glucose and [35S]sulfate, and with [35S]-sulfate only in human tissue. Both enzymes (2.6 x 10(-9) to 1.3 x 10(-6)M elastase and 8 x 10(-9) to 2.4 x 10(-6)M alkaline protease) released radiolabeled macromolecules in a concentration-dependent manner from the two preparations. Purified elastase, 1.3 x 10(-6)M, released radiolabeled macromolecules (delta 3H = +397 +/- 72%, delta 35S 225 +/- 40% over control, P less than 0.001) and periodic acid-Schiff- (PAS) reactive glycoconjugates (delta PAS = +4.1 +/- 0.96 micrograms/min or +102 +/- 20%; P less than 0.01) from cat trachea, as did alkaline protease, 2.4 x 10(-6)M (delta 3H = +356 +/- 57%, delta 35S = +176 +/- 25%, delta PAS = +7.5 +/- 1.3 micrograms/min or 194 +/- 36%, P less than 0.001). Increases in 3H exceeded those of 35S, suggesting surface epithelium as the main source of secretion. Inhibition of enzyme activity abolished secretory effects. Both enzymes also stimulated secretion from human bronchus (e.g., with elastase, 1.3 x 10(-6)M: delta 35S = +331 +/- 67%, delta PAS = +4.3 +/- 0.92 micrograms/min or +131 +/- 24%, P less than 0.001; with alkaline protease, 2.4 x 10(-6)M: delta 35S = +220 +/- 67%, delta PAS = +12.7 +/- 3.2 micrograms/min or +575 +/- 245%, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Dopamine promotes colonic mucus secretion through dopamine D5 receptor in rats

2019 ◽  
Vol 316 (3) ◽  
pp. C393-C403 ◽  
Author(s):  
Yun Li ◽  
Yue Zhang ◽  
Xiao-Li Zhang ◽  
Xiao-Yan Feng ◽  
Chen-Zhe Liu ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Colonic Mucosa ◽  
Periodic Acid ◽  
Mucus Secretion ◽  
Mucosal Barrier ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intracellular Camp ◽  
Periodic Acid Schiff ◽  
Dopamine Content

Dopamine regulates gastrointestinal mucosal barrier. Mucus plays important roles in the protection of intestinal mucosa. Here, the regulatory effect of dopamine on rat colonic mucus secretion was investigated. RT-PCR, immunofluorescence, Periodic Acid-Schiff reagent assay, Alcian blue-Periodic Acid-Schiff staining, and enzyme-linked immunosorbent assay were used to observe the expression of dopamine receptor and the direct effect of dopamine on the colonic mucus. Mice injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) destroying enteric dopamine (DA) neurons, rats microinjected with 6-hydroxydopamine (6-OHDA) into the bilateral substantia nigra damaging central dopaminergic neurons, and dopamine D5 receptor-downregulated transgenic mice were used to detect the effect of endogenous enteric dopamine or dopamine receptors on distal colonic mucus. Our results indicated that D5 immunoreactivity was widely distributed on the colonic goblet cells. Dopamine dose-dependently increased rat distal colonic mucus secretion in vitro. D1-like receptor antagonist SCH23390 inhibited dopamine (1 μΜ)-induced distal colonic mucus secretion. D1-like receptor agonist SKF38393 promoted mucin 2 (MUC2) secretion and increased the intracellular cAMP level of colonic mucosa. D5 receptor-downregulated transgenic mice showed a decreased colonic MUC2 content. MPTP-treated mice exhibited lower colonic dopamine content and decreased colonic mucus content. 6-OHDA rats had an increase in the dopamine content in colonic mucosa but decreases in the protein levels of D1 and D5 receptors and MUC2 content in the colonic mucosa. These findings reveal that dopamine is able to promote distal colonic mucus secretion through the D5 receptor, which provides important evidence to better understand the possible role of dopamine in the colonic mucosal barrier.

scholarly journals Histochemical properties of cartilage proteoglycans.

1983 ◽  
Vol 31 (1) ◽  
pp. 53-61 ◽  
Author(s):  
E Ippolito ◽  
V A Pedrini ◽  
A Pedrini-Mille
Keyword(s):  
Alcian Blue ◽  
Magnesium Chloride ◽  
Periodic Acid ◽  
Keratan Sulfate ◽  
Periodic Acid Schiff ◽  
Histological Sections ◽  
Connective Tissue Matrix ◽  
Cartilage Proteoglycans ◽  
Tissue Matrix

Proteoglycan interaction with alcian blue at different concentrations of magnesium chloride was studied both in vitro and in histological sections of paraffin-embedded tissues. Our experiments indicate that a) proteoglycans with different contents of chondroitin sulfate and keratan sulfate, prepared under nondegradative conditions, are not distinguishable on the basis of the critical electrolyte concentrations at which staining is abolished; b) the state of aggregation of proteoglycans only very slightly affects the alcian blue affinity of the macromolecules at different concentrations of magnesium chloride; c) the interaction of proteoglycans with other components of the connective tissue matrix is an important factor in determining the strength of binding of alcian blue to the polyanionic macromolecules in histological sections. These factors should be considered in interpreting histochemical data obtained by staining tissue sections with alcian blue at different concentrations of magnesium chloride. Proteoglycans, like glycosaminoglycans, are only weakly periodic acid-Schiff-positive.

scholarly journals Mitochondria-Targeted Antioxidant Mito-Tempo Protects Against Aldosterone-Induced Renal Injury In Vivo

2017 ◽  
Vol 44 (2) ◽  
pp. 741-750 ◽  
Author(s):  
Wei Ding ◽  
Tingyan Liu ◽  
Xiao Bi ◽  
Zhiling Zhang
Keyword(s):  
Electron Microscopy ◽  
Renal Function ◽  
Nlrp3 Inflammasome ◽  
Renal Injury ◽  
Periodic Acid ◽  
Inflammasome Activation ◽  
Periodic Acid Schiff ◽  
Nlrp3 Inflammasome Activation

Background/Aims: Growing evidence suggests mitochondrial dysfunction (MtD) and the Nlrp3 inflammasome play critical roles in chronic kidney disease (CKD) progression. We previously reported that Aldosterone (Aldo)-induced renal injury in vitro is directly caused by mitochondrial reactive oxygen species (mtROS)-mediated activation of the Nlrp3 inflammasome. Here we aimed to determine whether a mitochondria-targeted antioxidant (Mito-Tempo) could prevent Aldo-induced kidney damage in vivo. Methods: C57BL/6J mice were treated with Aldo and/or Mito-Tempo (or ethanol as a control) for 4 weeks. Renal injury was evaluated by Periodic Acid-Schiff reagent or Masson’s trichrome staining and electron microscopy. ROS were measured by DCFDA fluorescence and ELISA. MtD was determined by real-time PCR and electron microscopy. Activation of the Nlrp3 inflammasome and endoplasmic reticulum stress (ERS) was detected via western blot. Results: Compared with control mice, Aldo-infused mice showed impaired renal function, increased mtROS production and MtD, Nlrp3 inflammasome activation, and elevated ERS. We showed administration of Mito-Tempo significantly improved renal function and MtD, and reduced Nlrp3 inflammasome activation and ERS in vivo. Conclusion: Mitochondria-targeted antioxidants may attenuate Aldo-infused renal injury by inhibiting MtD, the Nlrp3 inflammasome, and ERS in vivo. Therefore, targeting mtROS might be an effective strategy for preventing CKD.

scholarly journals Production of capsular material by equine trophoblast transplanted into immunodeficient mice

Reproduction ◽  
2003 ◽  
pp. 855-863 ◽  
Author(s):  
A Albihn ◽  
RO Waelchli ◽  
J Samper ◽  
JG Oriol ◽  
BA Croy ◽  
...  
Keyword(s):  
Periodic Acid ◽  
Endometrial Biopsy ◽  
Specific Staining ◽  
Periodic Acid Schiff ◽  
In Vitro Techniques ◽  
Immunodeficient Mice ◽  
Xenogeneic Transplantation ◽  
Pas Staining ◽  
Capsular Material

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.

scholarly journals Differentiation of Human Umbilical Cord Lining Membrane-Derived Mesenchymal Stem Cells into Hepatocyte-Like Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Chinh Chung Doan ◽  
Thanh Long Le ◽  
Nghia Son Hoang ◽  
Dinh Nguyen Ky ◽  
Hoang Chuong Nguyen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Low Density Lipoprotein ◽  
Periodic Acid ◽  
Density Lipoprotein ◽  
Human Umbilical Cord ◽  
Periodic Acid Schiff ◽  
Differentiated Cells

Background. Mesenchymal stem cells (MSCs), isolated from bone marrow, adipose tissue, and umbilical cord tissue, have been known to differentiate into hepatocyte-like cells. MSCs can also be easily obtained from umbilical cord lining membrane (CLMSCs). CLMSCs are more primitive MSCs than those isolated from other tissue sources. Objectives. The aim of this study was to investigate the in vitro differentiation of CLMSCs into hepatocyte lineage. Materials and Methods. In this study, CLMSCs were isolated through a tissue attachment method. Cells were characterized for expression of MSC-specific markers and differentiation potency. CLMSCs were induced to differentiate into hepatocytes by a simple two-step protocol. Differentiated cells were examined for the expression of hepatocyte-specific markers and hepatocyte functions. Results. CLMSCs expressed MSC-specific markers and differentiated into adipocytes and osteoblasts. RT-PCR, real-time qRT-PCR, Western blot, and immunocytochemistry analyses demonstrated that differentiated CLMSCs, having hepatocyte-like morphology, expressed several liver-specific markers, such as ALB, AFP, CK18, and CK19, at both mRNA and protein levels following hepatocyte differentiation. Furthermore, periodic acid-Schiff staining and low-density lipoprotein (LDL) uptake assay showed that differentiated cells could store glycogen and uptake LDL. Conclusion. This study demonstrated that CLMSCs can differentiate into functional hepatocyte-like cells. CLMSCs can serve as a favorable cell source for tissue engineering in the treatment of liver disease.

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