scholarly journals Mucin-like glycoprotein secreted by cultured hamster tracheal epithelial cells. Biochemical and immunological characterization

1991 ◽  
Vol 277 (3) ◽  
pp. 713-718 ◽  
Author(s):  
R Wu ◽  
C G Plopper ◽  
P W Cheng
Keyword(s):  
Density Gradient ◽  
Influenza A ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Total Amino Acid ◽  
Tracheal Epithelial Cells ◽  
Cscl Density Gradient ◽  
Tracheal Epithelial ◽  
Chondroitin Ac Lyase

We isolated mucin-like glycoproteins from the conditioned medium of primary hamster tracheal epithelial (HTE) cell culture and characterized them biochemically and immunologically. These glycoproteins were purified on Sepharose CL-4B after Streptomyces hyaluronidase treatment and then by CsCl-density-gradient centrifugation in the presence of 4 M-guanidinium chloride. The purified glycoproteins were resistant to digestion by chondroitin AC lyase, heparinase, heparitinase and endo-N-acetylglucosaminidases A, D and H, but susceptible to endo-beta-galactosidase and keratanase. SDS/PAGE demonstrated no contamination by low-molecular-mass proteins. The purified glycoproteins showed a peak buoyant density of 1.56 g/ml in CsCl-density-gradient centrifugation, and contained 10% peptide and 90% carbohydrate by weight. Carbohydrates in these glycoproteins contained N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, sialic acid and a trace amount of mannose, but no uronic acid. Serine and threonine together accounted for 27% of the total amino acid residues. In addition, the mucin-like glycoproteins exhibited blood-group A and B activities, and very strong inhibitory activity for influenza A virus haemagglutination. With the use of the purified glycoprotein as an antigen, six monoclonal antibodies that stained mucus granules in hamster tracheal epithelium were obtained. We characterized the antibody produced by one of the clones, HM D46. We conclude that HTE cells cultured in the serum-free medium secrete a glycoprotein with physicochemical properties similar to those known in various airways mucins.

scholarly journals Mucins secreted by a transformed cell line derived from human tracheal gland cells1

1997 ◽  
Vol 326 (2) ◽  
pp. 431-437 ◽  
Author(s):  
Jean-Marc LO-GUIDICE ◽  
Marc D. MERTEN ◽  
Geneviève LAMBLIN ◽  
Nicole PORCHET ◽  
Marie-Christine HOUVENAGHEL ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Cell Line ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
High Molecular Mass ◽  
Cscl Density Gradient

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose® CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to β-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc α2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520–528]; they should be considered as having a mixed, both serous and mucous, phenotype.

scholarly journals The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage

1984 ◽  
Vol 221 (3) ◽  
pp. 637-644 ◽  
Author(s):  
P J Roughley ◽  
R J White ◽  
A R Poole ◽  
J S Mort
Keyword(s):  
Hyaluronic Acid ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Adult Human ◽  
Cscl Density Gradient ◽  
Proteoglycan Aggregates

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.

scholarly journals Isolation of different high-Mr mucin species from human whole saliva

1992 ◽  
Vol 283 (3) ◽  
pp. 807-811 ◽  
Author(s):  
E C I Veerman ◽  
P A M van den Keybus ◽  
M Valentijn-Benz ◽  
A V Nieuw Amerongen
Keyword(s):  
Monoclonal Antibodies ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Density Gradient Ultracentrifugation ◽  
Immunochemical Analysis ◽  
Guanidinium Chloride ◽  
Whole Saliva ◽  
Cscl Density Gradient

By using CsCl-density-gradient ultracentrifugation, two high-Mr mucin species were isolated from human whole saliva, having buoyant densities in 0.2 M-guanidinium chloride of approx. 1.56 g/ml (pool IA) and 1.48 g/ml (pool IIA). Analytical density-gradient centrifugation of submandibular, sublingual, labial and palatal saliva, followed by immunochemical analysis with anti-mucin monoclonal antibodies, indicated immunochemical and physicochemical similarities between the high-density mucins of pool IA and mucins from palatal salivary glands. Chemical analysis indicated that the putative palatal mucin was rich in sulphate, but poor in sialic acid. The lower-density mucins of pool IIA equated with the high-Mr mucins of submandibular-sublingual saliva, both immunochemically and physicochemically (buoyant density).

scholarly journals Interactions between different corneal proteoglycans

1978 ◽  
Vol 173 (3) ◽  
pp. 935-939 ◽  
Author(s):  
P Speziale ◽  
M S Speziale ◽  
L Galligani ◽  
C Balduini
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Original Sample ◽  
Chemical Analyses ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Cscl Density Gradient ◽  
Two Peaks ◽  
Fraction Density

Proteoglycans were extracted from bovine cornea with 4M-guanidinium chloride and purified by CsCl-density-gradient centrifugation. Under associative conditions two fractions were found: one capable of forming assemblies of high molecular weight and another lacking this property. The heavier fraction (density 1.59 g/ml) was eluted as a single retarded peak from Sepharose 2B, but on DEAE-Sephadex chromatography, gave two peaks: the first (eluted with 0.75 M-NaCl) contained mainly proteochondroitin sulphate and the second (eluted with 1.25 M-NaCl) mainly proteokeratan sulphate. Each of these proteoglycans was more retarded on Sepharose 2B than was the original sample from density-gradient centrifugation. Re-aggregation was obtained by recombination of the two fractions. The lighter fraction (density 1.44 g/ml), containing predominantly keratan sulphate chains, was eluted from DEAE-Sephadex as a single peak with 1.25 M-NaCl and was retarded on Sepharose 2B: this fraction was not able to form aggregates with proteochondroitin sulphate. Chemical analyses of the carbohydrate and protein moieties of the proteoglycans from DEAE-Sephadex confirmed that, in the cornea, different subunits are present with characteristic aggregation properties and hydrodynamic volumes.

scholarly journals Evidence that platelet density depends on the alpha-granule content in platelets

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 482-485 ◽  
Author(s):  
BA van Oost ◽  
AP Timmermans ◽  
JJ Sixma
Keyword(s):  
Density Distribution ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Platelet Rich Plasma ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Adenosine Diphosphate ◽  
Density Gradients

Abstract The relation between platelet buoyant density and beta-thromboglobulin (beta-TG), a marker for platelet alpha-granule content, was assessed by three independent approaches. (1) Platelets were separated on iso- osmolar discontinuous Stractan density gradients into five fractions, ranging in density from 1.061 g/ml to 1.091 g/ml (20 degrees C). The beta-TG content (mean +/- SD, n = 17) increased with the platelet density from 27.8 +/- 8.6 micrograms beta-TG/10(9) cells (20% less- dense platelets) up to 65.6 +/- 15.5 micrograms beta-TG/10(9) cells (15% most-dense platelets). (2) Activation of platelets in platelet- rich plasma with thrombin, adenosine diphosphate, collagen, or epinephrine resulted in a decreased density of the platelets. This was only seen when there was simultaneous secretion of beta-TG. (3) The less-dense and the more-dense platelet fractions, after isolation by density gradient centrifugation, were separately treated with thrombin. After complete degranulation, the density distribution of the originally less-dense and more-dense platelets were identical and were much narrower than the density distribution of resting platelets.

Transgressive, reiterative selection by continuous buoyant density gradient centrifugation of Dunaliella salina results in enhanced lipid and starch content

2015 ◽  
Vol 9 ◽  
pp. 194-203 ◽  
Author(s):  
Leyla T. Hathwaik ◽  
Doug Redelman ◽  
Vera Samburova ◽  
Barbara Zielinska ◽  
David K. Shintani ◽  
...  
Keyword(s):  
Density Gradient ◽  
Dunaliella Salina ◽  
Density Gradient Centrifugation ◽  
Starch Content ◽  
Gradient Centrifugation ◽  
Buoyant Density

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 Survival of density subpopulations of rabbit platelets: use of 51Cr-or 111In-labeled platelets to measure survival of least dense and most dense platelets concurrently

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 362-367 ◽  
Author(s):  
ML Rand ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Density Gradient ◽  
Total Population ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Specific Radioactivity ◽  
Rabbit Platelets

Abstract The origin of the density heterogeneity of platelets was studied by measuring the survival of density subpopulations of rabbit platelets separated by discontinuous Stractan density gradient centrifugation. When a total population of 51Cr-labeled platelets was injected into recipient rabbits, the relative specific radioactivity of the most dense platelets decreased rapidly. In contrast, that of the least dense platelets had not changed 24 hr after injection, and then decreased slowly. To distinguish between the possibilities that most dense platelets are cleared from the circulation more quickly than least dense platelets or that platelets decrease in density as they age in the circulation, the concurrent survival of least dense and most dense platelets, labeled with either 51Cr or 111In-labeled total platelet populations, determined concurrently in the same rabbits, were identical, calculated from 1 hr values as 100%. However, the 1-hr recovery of 111In-labeled platelets was slightly but significantly less than that of 51Cr-labeled platelets. Therefore, we studied the survival of 51Cr-labeled least dense and 111In-labeled most dense platelets as well as that of 111In-labeled least dense and 51Cr-labeled most dense platelets. Mean 1-hr recovery of least dense platelets, labeled with either isotope (78% +/- 7%, SD) was similar to that of most dense platelets, labeled with either isotope (77% +/- 8%; SD). Mean survival of least dense platelets was 47.3 +/- 18.7 hr (SD), which was significantly less than that of most dense platelets (76.1 +/- 21.6 hr; SD) (p less than 0.0025). These results indicate that platelets decrease in buoyant density as they age in the circulation and that most dense platelets are enriched in young platelets, and least dense in old. Thus, the events that affect platelets as they age in the circulation contribute to platelet density heterogeneity, although they may not be the sole cause of it.

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