scholarly journals Characterization and localization of the putative ‘link’ component in rat small-intestinal mucin

1987 ◽  
Vol 243 (3) ◽  
pp. 631-640 ◽  
Author(s):  
R E F Fahim ◽  
R D Specian ◽  
G G Forstner ◽  
J F Forstner
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Intestinal Lumen ◽  
Western Blots ◽  
Peptide Bonds ◽  
Disulphide Bonds ◽  
Purification Technique ◽  
Intestinal Mucin ◽  
Small Intestinal ◽  
Monospecific Antibodies ◽  
Link Component

Rat intestinal mucin is polymerized by a putative ‘link’ component of Mr 118,000 that can be released from the native mucin by thiol reduction [Fahim, Forstner & Forstner (1983) Biochem. J. 209, 117-124]. To confirm that this component is an integral part of the mucin and independent of the mucin purification technique, rat mucin was purified in the present study by three independent techniques. In all cases, the 118,000-Mr component was released after reduction. The 118 kDa band was electroeluted from SDS/polyacrylamide gels and its composition shown to resemble closely that of the link component of human intestinal mucin [Mantle, Forstner & Forstner (1984) Biochem. J. 224, 345-354]. Carbohydrates were present, including significant (10 mol/100 mol) amounts of mannose, suggesting the presence of N-linked oligosaccharides. Monospecific antibodies prepared against the rat 118,000-Mr component established its tissue localization in intestinal goblet cells. Mucins subjected to SDS/polyacrylamide-gel electrophoresis and Western blots using the same antibody, established that the link components of rat and human intestinal mucin are similar antigenically. Brief exposure (10 min) of native rat mucin to trypsin or Pronase (enzyme/mucin protein, 1:500, w/w) also released a 118,000-Mr component that reacted with the monospecific antibody. Thus the 118,000-Mr component is an integral part of the mucin and, although linked to large glycopeptides by disulphide bonds, this component also has proteinase-sensitive peptide bonds, presumably at terminal locations such that brief treatment with proteinases releases the molecule in a reasonably intact form. Under physiological conditions, therefore, one might expect that, after mucin is secreted into the intestinal lumen, luminal proteinases would rapidly remove the link component, thereby causing the mucin to depolymerize.

scholarly journals Antigenic and structural features of goblet-cell mucin of human small intestine

1984 ◽  
Vol 217 (1) ◽  
pp. 159-167 ◽  
Author(s):  
M Mantle ◽  
G G Forstner ◽  
J F Forstner
Keyword(s):  
Goblet Cell ◽  
Tertiary Structure ◽  
Solid Phase ◽  
Three Dimensional ◽  
Structural Features ◽  
Antigenic Determinants ◽  
Western Blots ◽  
Intestinal Mucin ◽  
Small Intestinal ◽  
Three Dimensional Configuration

With the use of a newly developed solid-phase radioimmunoassay method, the major antigenic determinants of human small-intestinal goblet-cell mucin were investigated and related to the overall tertiary structure of the mucin. Preliminary hapten inhibition studies with various oligosaccharides of known sequence and structure suggested that the determinants did not reside in carbohydrate. Exhaustive thiol reduction, however, almost abolished antigenicity, caused breakdown of the mucin into small heterogeneous glycopeptides, and liberated a ‘link’ peptide of Mr 118000. Western ‘blots’ of reduced mucin from polyacrylamide gels on to nitrocellulose sheets showed that a small amount of residual antigenicity remained in large-Mr glycopeptides (Mr greater than 200000). The ‘link’ peptide was not antigenic. Timed Pronase digestion of native mucin resulted in a progressive loss of antigenic determinants. Gel electrophoresis revealed that after 8h of digestion the 118000-Mr peptide had disappeared, whereas antigenicity, which was confined to large-Mr glycopeptides, was destroyed much more slowly with time (70% by 24h, 100% by 72h). Despite the loss of antigenicity, 72h-Pronase-digested glycopeptides retained all of the carbohydrate of the native mucin. Therefore the antibody to human small-intestinal mucin appears to recognize a ‘naked’ (non-glycosylated and Pronase-susceptible) peptide region(s) of mucin glycopeptides. For full antigenicity, however, disulphide bonds are required to stabilize a specific three-dimensional configuration of the ‘naked’ region.

Characterization of an endogenous quail intestinal lectin

1990 ◽  
Vol 68 (2) ◽  
pp. 471-475 ◽  
Author(s):  
Rixun Fang ◽  
Howard Ceri
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Immunohistochemical Localization ◽  
Immunoperoxidase Staining ◽  
Lectin Activity ◽  
Western Blots ◽  
Intestinal Mucin ◽  
Simple Sugar

Soluble extracts of quail intestine scrapings contain a lectin activity specific for chicken and rabbit trypsinized, glutaraldehyde-fixed erythrocytes. The lectin displayed a specificity for the simple sugar haptens lactose and galactose and for mucin. Quail lectin was purified by affinity chromatography on either asialofetuin- or mucin-Sepharose, followed by DEAE-Sepharose chromatography, and demonstrated an apparent molecular weight of 14 500 on sodium dodecyl sulfate – polyacrylamide gel electrophoresis and a pi of 6.2 upon isoelectric focusing. Immunohistochemical localization of this lectin in the intestine was carried out using polyclonal antibody raised in rabbits and tested for specificity in Western blots. Immunoperoxidase staining for quail lectin showed the lectin to be prominent in secretions at the mucosal surface and in goblet cells.Key words: endogenous lectin, intestinal, mucin, goblet cells.

scholarly journals Effects of hydrogen peroxide, mild trypsin digestion and partial reduction on rat intestinal mucin and its disulphide-bound 118 kDa glycoprotein

1991 ◽  
Vol 274 (3) ◽  
pp. 679-685 ◽  
Author(s):  
M Mantle
Keyword(s):  
Gel Electrophoresis ◽  
High Molecular Mass ◽  
Void Volume ◽  
Elution Profile ◽  
Partial Reduction ◽  
Progressive Loss ◽  
Disulphide Bonds ◽  
Intestinal Mucin ◽  
Link Component

The role of the disulphide-bound 118 kDa glycoprotein of rat intestinal mucin is unknown, although it has been proposed to serve as a ‘link’ component for the mucin monomers. The present studies investigated release or destruction of the 118 kDa glycoprotein (monitored by gel electrophoresis and Western-blot analysis) during progressive breakdown of the mucin polymer (assessed by Sepharose 2B chromatography). H2O2 gradually destroyed the 118 kDa glycoprotein and dissociated the mucin polymer into components of similar size to the monomers. After 3 h, mucin samples contained almost no 118 kDa glycoprotein or its breakdown products, but 50% of the mucin was still eluted in the void volume of a Sepharose 2B column. Although mild trypsinolysis had little effect on the Sepharose 2B elution profile of the mucin, the 118 kDa glycoprotein was completely cleaved into 54-56 kDa and 60-66 kDa fragments which remained disulphide-bound to the high-molecular-mass mucin. Increasing levels of thiol reduction resulted in progressive loss of disulphide bonds, release of the 118 kDa glycoprotein and depolymerization of the mucin. Although approx. 40% of the mucin in partially reduced samples was recovered in the Sepharose 2B void volume, this material contained no 118 kDa glycoprotein and apparently consisted of disulphide-bound mucin monomers. Thus the 118 kDa glycoprotein may be destroyed by H2O2, extensively cleaved by trypsin or released by reduction without completely dissociating the mucin into monomers. Therefore the 118 kDa glycoprotein may not function as a ‘link’ component for all of the mucin monomers in the native polymer.

Use of shape-from-shading to estimate three-dimensional architecture in the small intestinal lumen of celiac and control patients

2013 ◽  
Vol 111 (3) ◽  
pp. 676-684 ◽  
Author(s):  
Edward J. Ciaccio ◽  
Christina A. Tennyson ◽  
Govind Bhagat ◽  
Suzanne K. Lewis ◽  
Peter H.R. Green
Keyword(s):  
Three Dimensional ◽  
Shape From Shading ◽  
Intestinal Lumen ◽  
Small Intestinal ◽  
And Control

Phytates reduce uptake of leucine and glutamate but not lysine and glucose from the intestinal lumen of chickens: Short communication

2008 ◽  
Vol 56 (4) ◽  
pp. 511-514 ◽  
Author(s):  
Edward Onyango ◽  
Elikplimi Asem ◽  
Olayiwola Adeola
Keyword(s):  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Short Communication ◽  
Intestinal Lumen ◽  
Depressive Effect ◽  
Leucine Uptake ◽  
Small Intestinal

An investigation into the influence of phytates on the in situ absorption of amino acids (lysine, glutamate and leucine) and glucose from the intestinal lumen of 3-week-old chickens was carried out. Birds were anaesthetised and the intestines exteriorised. Uptake of 5 mM of each nutrient over a 4-min period was measured in the presence of four phytate concentrations (0, 50, 250 and 500 mM). Five birds were used for each nutrient at each concentration of phytate tested. Leucine uptake decreased linearly (P < 0.001) and that of glutamate showed a tendency to decrease (P = 0.055) as the phytate concentration increased. Absorption of lysine and glucose were unaffected by the presence of phytate. In conclusion, phytate in the small intestinal lumen exerted a depressive effect on the absorption of specific free amino acids from the lumen. Its depressive effect was greatest for leucine followed by glutamate, and phytate had little effect on the absorption of lysine.

scholarly journals Dietary ascorbic acid lowers the concentration of soluble copper in the small intestinal lumen of rats

1994 ◽  
Vol 71 (5) ◽  
pp. 701-707 ◽  
Author(s):  
G. J. Van Den Berg ◽  
S. YU ◽  
A. G. Lemmens ◽  
A. C. Beynen
Keyword(s):  
Ascorbic Acid ◽  
Small Intestine ◽  
Liquid Phase ◽  
Inhibitory Effect ◽  
Male Rats ◽  
Intestinal Lumen ◽  
Distal Intestine ◽  
Small Intestinal ◽  
Ascorbic Acid Supplementation

We tested the hypothesis that ascorbic acid in the diet of rats lowers the concentration of soluble Cu in the small intestine, causing a decrease in apparent Cu absorption. Male rats were fed on diets adequate in Cu (5 mg Cu/kg) without or with 10 g ascorbic acid/kg. The diet with ascorbic acid was fed for either 6 or 42 d. Ascorbic acid depressed tissue Cu concentrations after a feeding period of 42, but not after 6 d. Dietary ascorbic acid lowered apparent Cu absorption after 6, but not after 42 d. The lowering of tissue Cu concentrations after long-term ascorbic acid feeding may have increased the efficiency of Cu absorption, and thus counteracted the inhibitory effect of ascorbic acid. Dietary ascorbic acid caused a significant decrease in the Cu concentrations in the liquid phase of both the proximal and distal parts of the small intestinal lumen. This effect was due to both a decrease in the amount of Cu in the liquid digesta and an increase in the volume of the liquid phase; only the latter effect for the distal intestine was statistically significant. We conclude that ascorbic acid supplementation lowers Cu absorption by decreasing the concentration of soluble Cu in the small intestine.

The synthesis of type X collagen by bovine and human growth-plate chondrocytes

1991 ◽  
Vol 99 (3) ◽  
pp. 641-649 ◽  
Author(s):  
A. Marriott ◽  
S. Ayad ◽  
M.E. Grant
Keyword(s):  
Growth Plate ◽  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Growth ◽  
Type I ◽  
Collagen Gels ◽  
Type X Collagen ◽  
Growth Plate Chondrocytes ◽  
Growth Plate Cartilage ◽  
Disulphide Bonds

Chondrocytes were isolated from bovine growth-plate cartilage and cultured within type I collagen gels. A major collagen with chains of Mr 59,000, decreasing to 47,000 on pepsinization, was synthesized and identified as type X collagen. This collagen was cleaved at two sites by mammalian collagenase, resulting in a major triple-helical fragment with chains of Mr 32,000. The species of Mr 59,000, 47,000 and 32,000 were not detected by SDS-polyacrylamide gel electrophoresis before reduction, indicating the presence of disulphide bonds within the triple helix. In contrast, similar biosynthetic studies with human growth-plate cartilage in organ culture, indicated that human type X collagen does not contain disulphide bonds. A polyclonal antiserum was raised to bovine type X collagen and used in immunolocalization studies to provide direct evidence for the association of type X collagen with the hypertrophic chondrocytes in both bovine and human growth plates during development.

Isolation, Cultivation, and Characterization of Borrelia burgdorferi from Rodents and Ticks in the Charleston Area of South Carolina

2000 ◽  
Vol 38 (1) ◽  
pp. 120-124
Author(s):  
J. H. Oliver ◽  
K. L. Clark ◽  
F. W. Chandler ◽  
L. Tao ◽  
A. M. James ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Borrelia Burgdorferi ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Cotton Rat ◽  
Cotton Mouse ◽  
B 31

ABSTRACT Twenty-eight Borrelia burgdorferi isolates from the Charleston, S.C., area are described. This represents the first report and characterization of the Lyme disease spirochete from that state. The isolates were obtained from December 1994 through December 1995 from the tick Ixodes scapularis , collected from vegetation, and from the rodents Peromyscus gossypinus (cotton mouse), Neotoma floridana (eastern wood rat), and Sigmodon hispidus (cotton rat). All isolates were screened immunologically by indirect immunofluorescence with monoclonal antibodies to B. burgdorferi -specific outer surface protein A (OspA) (antibodies H5332 and H3TS) and B. burgdorferi -specific OspB (antibodies H6831 and H614), a Borrelia (genus)-specific antiflagellin antibody (H9724), Borrelia hermsii -specific antibodies (H9826 and H4825), and two polyclonal antibodies (one to Borrelia species and another to B. burgdorferi ). Six of the isolates were analyzed by exposing Western blots to monoclonal antibodies H5332, H3TS, H6831, and H9724. All isolates were also analyzed by PCR with five pairs of primers known to amplify selected DNA target sequences specifically reported to be present in the reference strain, B. burgdorferi B-31. The protein profiles of six of the isolates (two from ticks, one from a cotton mouse, two from wood rats, and one from a cotton rat) also were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the 28 Charleston isolates are B. burgdorferi sensu stricto based on their similarities to the B. burgdorferi B-31 reference strain.

scholarly journals TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

2020 ◽  
Vol 5 (47) ◽  
pp. eabc3582 ◽  
Author(s):  
Ruochen Zang ◽  
Maria Florencia Gomez Castro ◽  
Broc T. McCune ◽  
Qiru Zeng ◽  
Paul W. Rothlauf ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Gastrointestinal Symptoms ◽  
Host Cells ◽  
Productive Infection ◽  
Intestinal Lumen ◽  
Fecal Shedding ◽  
Oral Transmission ◽  
Stool Specimens ◽  
Small Intestinal ◽  
Intestinal Enterocytes

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19 patients. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

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