scholarly journals Mucus glycoproteins from pig gastric mucosa: identification of different mucin populations from the surface epithelium

1997 ◽  
Vol 326 (3) ◽  
pp. 903-910 ◽  
Author(s):  
Henrik NORDMAN ◽  
Julia R. DAVIES ◽  
Annkatrin HERRMANN ◽  
Niclas G. KARLSSON ◽  
Gunnar C. HANSSON ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Buoyant Density ◽  
High Density ◽  
Surface Epithelium ◽  
Low Density ◽  
Guanidinium Chloride ◽  
Gastric Mucus ◽  
High Iron ◽  
Cardiac Region ◽  
Ion Exchange Hplc

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

scholarly journals Mucus glycoproteins from pig gastric mucosa: different mucins are produced by the surface epithelium and the glands

1998 ◽  
Vol 331 (3) ◽  
pp. 687-694 ◽  
Author(s):  
Henrik NORDMAN ◽  
Julia R. DAVIES ◽  
Ingemar CARLSTEDT
Keyword(s):  
Gastric Mucosa ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Surface Epithelium ◽  
Gel Chromatography ◽  
Glandular Tissue ◽  
Cardiac Region ◽  
Ion Exchange Hplc ◽  
Density Population ◽  
Mucus Glycoproteins

An antibody (PGM2B) recognizing a pig gastric-mucin apoprotein reacts with the surface epithelium of pig gastric mucosa. Virtually no reactivity was observed over the mucin-producing cells in the glands, which were recognized by the GlcNAc-selective Griffonia simplicifoliaII (GSA-II) lectin. Mucins from the glandular tissue of the cardiac region, corpus and antrum were purified using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. In the cardiac region, two major mucin populations at 1.5 and 1.4 g/ml were identified. The high-density population reacted preferentially with the PGM2B antibody and resembled mucins from the surface epithelium of this region, whereas the low-density population reacted strongly with the GSA-II lectin and appeared to originate from the glands. In the glandular tissue of corpus, a component with strong GSA-II lectin reactivity, which was distinctly different from the surface mucins from this region, was found at 1.4 g/ml, thus resembling the gland component from the cardiac region. Mucins from antrum glandular tissue contained at least two GSA-II lectin-reactive populations banding at 1.5 and 1.4 g/ml, respectively. Gland mucins from all regions were large oligomeric glycoproteins and heterogeneous with respect to charge properties, as shown by using rate-zonal centrifugation and ion-exchange HPLC, respectively. Gel chromatography of mucin glycopeptides showed that gland mucins from antrum and corpus contained significantly longer glycosylated domains than those from the surface mucosa. Thus, mucins from pig gastric glandular tissue comprise a number of large and oligomeric glycoproteins that differ from those from the surface epithelium in buoyant density, apoprotein structure and carbohydrate substitution.

scholarly journals Mucus glycoproteins in bovine trachea: identification of the major mucin populations in respiratory secretions and investigation of their tissue origins

1997 ◽  
Vol 321 (1) ◽  
pp. 117-124 ◽  
Author(s):  
Hans W. HOVENBERG ◽  
Ingemar CARLSTEDT ◽  
Julia R. DAVIES
Keyword(s):  
Gradient Centrifugation ◽  
Complex Mixture ◽  
Goblet Cells ◽  
High Density ◽  
Surface Epithelium ◽  
Low Density ◽  
Submucosal Glands ◽  
Mucus Gel ◽  
Soluble Species ◽  
Gel Phase

Bovine respiratory secretions were separated into gel and sol phases to allow the identification of the gel-forming mucins. Mucins were subsequently isolated from the surface epithelium and submucosal tissue to investigate the tissue origins of the species in the secretions. Density-gradient centrifugation revealed ‘high-density’ and ‘low-density’ mucins in the gel phase of the secretions. The ‘high-density’ mucins were large, composed of subunits joined by disulphide bonds and contained two highly glycosylated domains of apparently different lengths, whereas the ‘low-density’ mucins were smaller and monomeric. The sol also contained both ‘high-density’ and ‘low-density’ species. A ‘high-density’ mucin similar to that in the gel was isolated from the surface epithelium, suggesting that the goblet cells produce large, gel-forming mucins. A second ‘high-density’ species was released from the submucosal tissue after reduction/alkylation, indicating that large mucins from the submucosal glands may also be a component of the mucus gel. In addition, two small, ‘low-density’ mucins were obtained from the submucosal tissue. One species was associated with the gel phase but was also present in the sol, whereas the other was present only in the sol. Bovine respiratory-tract secretions thus comprise a complex mixture of large gel-forming mucins originating from the goblet cells and submucosal glands, and smaller ‘soluble’ species from the submucosal glands which may interact with the gel.

Human Platelet Age Correlates With Buoyant Density, But Not With Size

1981 ◽  
Author(s):  
D Mezzano ◽  
P Catalano ◽  
K Hwang ◽  
R H Aster
Keyword(s):  
Life Span ◽  
Human Platelet ◽  
Buoyant Density ◽  
Specific Radioactivity ◽  
Normal Subjects ◽  
High Density ◽  
Low Density ◽  
Platelet Size ◽  
Post Infusion

Following infusion of 51Cr-labelled autologous platelets into normal subjects, high density (HD) and low density (LD) platelet cohorts were isolated by centrifugation in isosmotic arabino- galactan (Stractan) . Specific radioactivity (SA) of LD platelets declined rapidly post-infusion (T1/2 =1.8 days) but SA of HD platelets remained constant or increased over a 3-4 day period and gradually declined for 6-7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high density albumin were labelled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (p < .02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33 days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (p < .05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets = 7.57 μ3, LD platelets = 6.87 μ3, 0.05 < p < 0.01).These findings imply that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, SA of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, unlabelled platelets; SA of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions

scholarly journals MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage

1998 ◽  
Vol 334 (3) ◽  
pp. 685-693 ◽  
Author(s):  
Claes WICKSTRÖM ◽  
Julia R. DAVIES ◽  
Gitte V. ERIKSEN ◽  
Enno C. I. VEERMAN ◽  
Ingemar CARLSTEDT
Keyword(s):  
Respiratory Tract ◽  
Gradient Centrifugation ◽  
Guanidinium Chloride ◽  
Charge Densities ◽  
Terminal Domain ◽  
Disulphide Bonds ◽  
Whole Saliva ◽  
Submucosal Glands ◽  
Before And After ◽  
Ion Exchange Hplc

Mucins from human whole saliva, as well as from respiratory- and cervical-tract secretions, were subjected to density-gradient centrifugation in CsCl/0.5 M guanidinium chloride. A polydisperse population of MUC5B mucins was demonstrated in all samples using anti-peptide antisera (LUM5B-2, LUM5B-3 and LUM5B-4) raised against sequences within the MUC5B mucin. The sequences recognized by the LUM5B-2 and LUM5B-3 antisera are located within the domains flanking the highly glycosylated regions of MUC5B, and reduction increased the reactivity with these antibodies, suggesting that the epitopes are partially shielded and that these regions are folded and stabilized by disulphide bonds. Rate-zonal centrifugation before and after reduction showed MUC5B to be a large oligomeric mucin composed of disulphide-linked subunits. In saliva and respiratory-tract secretions, populations of MUC5B mucins with different charge densities were identified by ion-exchange HPLC, suggesting the presence of MUC5B ‘glycoforms ’. In trachea, the F2 monoclonal antibody against the sulpho-Lewis C structure reacted preferentially with the later-to-be-eluted populations. An antibody (LUM5B-4) recognizing a sequence in the C-terminal domain of MUC5B identified, after reduction, the mucin subunits as well as smaller fragments, suggesting that some of the MUC5B mucins are cleaved within the C-terminal domain. Immunohistochemistry revealed that MUC5B is produced by cells dispersed throughout the human submandibular and sublingual glands, in the airway submucosal glands as well as the goblet cells, and in the epithelium and glands of the endocervix. The F2 antibody stained a subpopulation of the MUC5B-producing cells in the airway submucosal glands, suggesting that different cells may produce different glycoforms of MUC5B in this tissue.

scholarly journals Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural ‘architecture’

1991 ◽  
Vol 276 (3) ◽  
pp. 667-675 ◽  
Author(s):  
D J Thornton ◽  
J K Sheehan ◽  
H Lindgren ◽  
I Carlstedt
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Gel Phase ◽  
Mucus Glycoproteins

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].

scholarly journals Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone

1989 ◽  
Vol 260 (3) ◽  
pp. 717-723 ◽  
Author(s):  
J Dekker ◽  
W M O Van Beurden-Lamers ◽  
A Oprins ◽  
G J Strous
Keyword(s):  
Buoyant Density ◽  
Gastric Mucin ◽  
Proteolytic Digestion ◽  
Guanidinium Chloride ◽  
Protein Backbone ◽  
Gastric Mucus ◽  
Electron Microscopic ◽  
Mucus Glycoprotein ◽  
Cscl Density Gradient ◽  
The Individual

We isolated monomeric gastric mucus glycoprotein from the rat stomach by applying three successive CsCl-density-gradient steps in the continuous presence of guanidinium chloride. The rat gastric mucin was pure as compared with mucin isolated without the chaotropic reagent. In addition, the presence of guanidinium chloride resulted in a better preservation of the protein moiety. The purified mucin was fractionated according to buoyant density and chemically radiolabelled on tyrosine or cysteine residues and digested with specific proteinases. Analysis of mucin fractions of various densities gave identical peptide patterns, suggesting that the fractions contain a common protein backbone. Electron-microscopic images of the individual mucin molecules were recorded using rotary shadowing. They showed large filamentous molecules with a mean length of 208 nm that, after proteolytic digestion, yielded glycopeptides with a mean length of 149 nm. Heterogeneity in buoyant density and electrophoretic mobility is located in this large glycopeptide which remains after proteolytic digestion. Metabolic labelling of the mucin with [35 S]sulphate and [3H]galactose, followed by purification and proteolytic digestion, revealed that this glycopeptide accounts for most of the mass and contains relatively little protein, but probably all the oligosaccharides and sulphate. As this protein part is masked by the oligosaccharides, detailed study by the methods described was not possible. The results indicate that rat gastric mucin is homogeneous in a major part of the protein backbone and that the heterogeneity of the molecule originates most likely from differences in sulphate and/or sugar composition.

scholarly journals Gastric MUC5AC and MUC6 are large oligomeric mucins that differ in size, glycosylation and tissue distribution

2002 ◽  
Vol 364 (1) ◽  
pp. 191-200 ◽  
Author(s):  
Henrik NORDMAN ◽  
Julia R. DAVIES ◽  
Gert LINDELL ◽  
Carme de BOLÓS ◽  
Francisco REAL ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Gradient Centrifugation ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Repeat Sequence ◽  
Surface Epithelium ◽  
Lewis Antigen ◽  
Gland Tissue ◽  
Before And After ◽  
Ion Exchange Hplc

Gastric MUC5AC and MUC6 mucins were studied using polyclonal antibodies. Immunohistochemistry showed MUC5AC to originate from the surface epithelium, whereas MUC6 was produced by the glands. Mucins from the surface epithelium or glands of corpus and antrum were purified using CsCl/4M guanidinium chloride density-gradient centrifugation. MUC5AC appeared as two distinct populations at 1.4 and 1.3g/ml, whereas MUC6, which was enriched in the gland tissue, appeared at 1.45g/ml. Reactivity with antibodies against the Leb structure (where Le represents the Lewis antigen) followed the MUC5AC distribution, whereas antibodies against the Ley structure and reactivity with the GlcNAc-selective Solanum tuberosum lectin coincided with MUC6, suggesting that the two mucins are glycosylated differently. Rate-zonal centrifugation of whole mucins and reduced subunits showed that both gastric MUC5AC and MUC6 are oligomeric glycoproteins composed of disulphide-bond linked subunits and that oligomeric MUC5AC was apparently smaller than MUC6. A heterogeneous population of ‘low-density’ MUC5AC mucins, which were smaller than the ‘high-density’ ones both before and after reduction, reacted with an antibody against a variable number tandem repeat sequence within MUC5AC, suggesting that they represent precursor forms of this mucin. Following ion-exchange HPLC, both MUC5AC and MUC6 appeared as several distinct populations, probably corresponding to ‘glycoforms’ of the mucins, the most highly charged of which were found in the gland tissue.

scholarly journals Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells

1996 ◽  
Vol 318 (1) ◽  
pp. 319-324 ◽  
Author(s):  
Hans W HOVENBERG ◽  
Julia R DAVIES ◽  
Ingemar CARLSTEDT
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Goblet Cells ◽  
Major Product ◽  
Surface Epithelium ◽  
Epithelial Surface ◽  
Predominant Species ◽  
Submucosal Glands ◽  
Ion Exchange Hplc ◽  
Human Trachea

Mucins were extracted from the epithelial surface and the submucosal tissue of human trachea in order to enrich glycoproteins from the goblet cells and the submucosal glands respectively. The macromolecules were purified using density-gradient centrifugation, and the presence of the MUC5AC mucin was investigated using an antiserum raised against a synthetic peptide based on the sequence of the MUC5AC apoprotein. Mucins from the surface epithelium showed a higher reactivity with the antiserum relative to carbohydrate than those from the submucosa, and ion-exchange HPLC of reduced subunits revealed the presence of two distinct mucin populations in the samples. The predominant species from the surface epithelium was more acidic than the major population from the submucosa and showed a strong reactivity with the anti-MUC5AC antiserum. In contrast, the major portion of the submucosal mucins were less acidic and showed no MUC5AC reactivity, although a more acidic population did react with the antibody. Rate-zonal centrifugation showed that the MUC5AC mucin from the surface epithelium is smaller than the major submucosal mucin, and that both are composed of subunits. Immunolocalization confirmed that the MUC5AC mucin from human trachea originates from the goblet cells and that this glycoprotein is not a major product of the submucosal glands.

scholarly journals Membrane Localization of Motility, Signaling, and Polyketide Synthetase Proteins in Myxococcus xanthus

2003 ◽  
Vol 185 (17) ◽  
pp. 5066-5075 ◽  
Author(s):  
Vesna Simunovic ◽  
Frank C. Gherardini ◽  
Lawrence J. Shimkets
Keyword(s):  
Protein Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Myxococcus Xanthus ◽  
Buoyant Density ◽  
High Density ◽  
Putative Hybrid ◽  
Low Density ◽  
Cellular Motility ◽  
Density Fraction ◽  
Density Fractions

ABSTRACT Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm−3). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm−3) and low buoyant density (1.169 to 1.171 g cm−3). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm−3) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.

scholarly journals Kinetic study of T lymphocytes after sensitization against soluble antigen. III. Potentiation and suppression of the PHA response by antigen-activated lymphocytes of low density.

1975 ◽  
Vol 142 (4) ◽  
pp. 1017-1022 ◽  
Author(s):  
J A Bash ◽  
H G Durkin ◽  
B H Waksman
Keyword(s):  
Buoyant Density ◽  
High Density ◽  
Soluble Antigen ◽  
Low Density ◽  
Constant Number ◽  
Activated Lymphocytes ◽  
Density Fractions ◽  
Lymph Node Cells ◽  
Two Populations ◽  
Pha Response

Lymph node cells of ovalbumin-sensitized rats were separated on the basis of buoyant density into fractions reciprocally enriched in cells responsive to ovalbumin or phytohemagglutinin (PHA). Recombination of high density and low density fractions in varying proportions resulted in potentiation or suppression of the DNA synthetic response to PHA in culture. The response of cultures containing equal numbers of high and low density cells was markedly greater than the sum of the two populations stimulated separately. However, when decreasing numbers of low density cells were cultured with a constant number of high density cells, profound suppression was observed.

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