Cubilin and megalin expression and their interaction in the rat intestine: effect of thyroidectomy

2001 ◽  
Vol 281 (5) ◽  
pp. E900-E907 ◽  
Author(s):  
Raghunatha R. Yammani ◽  
Shakuntla Seetharam ◽  
Bellur Seetharam
Keyword(s):  
Brush Border ◽  
Intrinsic Factor ◽  
Immunoblot Analysis ◽  
Rat Intestine ◽  
Complement Components ◽  
Protein Levels ◽  
Intestinal Brush Border ◽  
Molecular Masses ◽  
Epidermal Growth

Cubilin is a 460-kDa multipurpose, multidomain receptor that contains an NH2-terminal 110-residue segment followed by 8 epidermal growth factor (EGF)-like repeats and a contiguous stretch (representing nearly 88% of its mass) of 27 CUB (initially found in complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domains. Cubilin binds to intrinsic factor (IF)-cobalamin (cbl, vitamin B12) complex and promotes the ileal transport of cbl. The 460-kDa form of cubilin is the predominant form present in the apical brush-border membranes of rat intestine, kidney, and yolk sac, but a 230-kDa form of cubilin is also noted in the intestinal membranes. In thyroidectomized (TDX) rats, levels of intestinal brush-border IF-[57Co]-labeled cbl binding, 460-kDa cubilin protein levels and tissue (kidney) accumulation of cbl were reduced by ∼70%. Immunoblot analysis using cubilin antiserum of intestinal total membranes from TDX rats revealed cubilin fragments with molecular masses of 200 and 300 kDa. Both of these bands, along with the 230-kDa band detected in the total membranes of control rats and unlike the 460-kDa form, failed to react with antiserum to EGF. Mucosal membrane cubilin associated with megalin was reduced from ∼12% in control to ∼4% in TDX rats, and this decreased association was not due to altered megalin levels. Thyroxine treatment of TDX rats resulted in reversal of all of these effects, including an increase to nearly 24% of cubilin associated with megalin. In vitro, megalin binding to cubilin occurred with the NH2-terminal region that contained the EGF-like repeats and CUB domains 1 and 2 but not with a downstream region that contained CUB domains 2–10. These studies indicate that thyroxine deficiency in rats results in decreased uptake and tissue accumulation of cbl caused mainly by destabilization and deficit of cubilin in the intestinal brush border.

Divalent cations and vitamin B12 uptake by intestinal brush-border membrane vesicles

1980 ◽  
Vol 239 (6) ◽  
pp. G452-G456
Author(s):  
R. C. Beesley ◽  
C. D. Bacheller
Keyword(s):  
Vitamin B12 ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Divalent Cations ◽  
Intrinsic Factor ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Tetraacetic Acid ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border

Brush-border membrane vesicles from hamster intestine were employed to investigate uptake (binding) of vitamin B12 (B12). Ileal vesicles took up 25 times more B12 than did jejunal vesicles. Uptake of B12 by ileal vesicles was dependent on intrinsic factor (IF) and required Ca2+. Increasing the Ca2+ concentration caused an increase in uptake of B12 reaching a maximum at approximately 8 mM Ca2+. At high Ca2+ concentrations, 6–8 mM, Mg2+ had little effect on uptake of B12. At low Ca2+ concentrations, up to 2 mM, Mg2+ stimulated B12 uptake. Mg2+, Mn2+, and, to a lesser extent, Sr2+ stimulated Ca2+-dependent B12 uptake, but Zn2+, Ba2+, Na+, K+, and La3+ did not. B12 was apparently not metabolized and was bound as IF-B12 complex, which could be removed with (ethylenedinitrilo)tetraacetic acid (EDTA). Our results suggest that two types of divalent cation reactive sites are involved in binding of IF-B12. One is Ca2+ specific. The other is less specific reacting with Mg2+, Mn2+, Sr2+, and perhaps Ca2+ itself, thereby stimulating Ca2+-dependent binding of IF-B12 to its ileal receptor.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

1991 ◽  
Vol 69 (8) ◽  
pp. 499-508 ◽  
Author(s):  
Andrea G. Bodnar ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Peroxisome Proliferator ◽  
Peroxisomal Membrane ◽  
Molecular Masses ◽  
Fold Induction ◽  
Membrane Bound ◽  
Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

Differential calmodulin binding to three myosin-1 isoforms from liver

1994 ◽  
Vol 107 (8) ◽  
pp. 2279-2284 ◽  
Author(s):  
L.M. Coluccio
Keyword(s):  
Sequence Analysis ◽  
Molecular Mass ◽  
Gene Product ◽  
Brush Border ◽  
Peptide Sequence ◽  
Purification And Characterization ◽  
Calmodulin Binding ◽  
Intestinal Brush Border ◽  
Molecular Masses ◽  
Liver Homogenates

We have previously purified and characterized two myosin-1 isoforms from rat liver (molecular masses 130 kDa and 110 kDa; L. M. Coluccio and C. Conaty (1993) Cell Motil. Cytoskel. 24, 189–199). Here, we describe the purification and characterization from liver of a third myosin-1 (molecular mass 105 kDa) and determine the number of calmodulin molecules associated with each of these three myosin-1 isoforms. The 105 kDa polypeptide, solubilized from liver homogenates with the addition of ATP, co-sediments with F-actin, co-purifies with calmodulin, and binds calmodulin in the presence of EGTA. Antibodies directed against chicken intestinal brush border myosin-1 cross-react with the 105 kDa polypeptide on immunoblots. Partial peptide sequence analysis indicates that the polypeptide corresponds with an MM1 gamma gene product that represents a myosin-1 isoform cloned from mouse brain (Sherr et al. (1993) J. Cell Biol. 120, 1405–1416). A comparison of calmodulin binding to the now three isolated forms of myosin-1 in liver shows that in solution the 105 kDa and 110 kDa polypeptides bind two molecules of calmodulin each whereas the 130 kDa binds six molecules of calmodulin.

Some species differences in the intrinsic factor stimulation of B12 uptake by small intestine in vitro

1959 ◽  
Vol 197 (4) ◽  
pp. 926-928 ◽  
Author(s):  
T. Hastings Wilson ◽  
Elliott W. Strauss
Keyword(s):  
Small Intestine ◽  
Guinea Pig ◽  
Species Differences ◽  
Intrinsic Factor ◽  
Vitamin B ◽  
Guinea Pig Ileum ◽  
Rat Intestine ◽  
Intrinsic Factors ◽  
Stimulation Of

Sacs of everted small intestine from a variety of animals were incubated in bicarbonate-saline containing vitamin B12 with and without intrinsic factor (IF). B12 uptake by rat intestine was stimulated only by its own intrinsic factor. Guinea pig ileum responded to all intrinsic factors tested (guinea pig, rat, hog, hamster, human being and rabbit). The intestines of hamster and rabbit were intermediate in specificity, responding to some, but not all, of the IF preparations. Species differences occur in both the intestine and intrinsic factor preparations. The guinea pig ileum was suggested as a possible assay for both hog and human IF.

Hemileaflet susceptibility to oxidative damage in the intestinal brush-border membrane

1995 ◽  
Vol 268 (2) ◽  
pp. G260-G269
Author(s):  
D. Jourd'heuil ◽  
S. Mehta ◽  
J. B. Meddings
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Peroxyl Radical ◽  
Tissue Injury ◽  
Biological Membranes ◽  
First Order ◽  
Intestinal Brush Border Membrane ◽  
Outer Leaflet ◽  
Intestinal Brush Border

Oxidation of biological membranes is characteristic of many types of tissue injury, including those observed with inflammatory bowel disease. The lipid compositions of the inner and outer leaflets of biological membranes differ significantly, making one leaflet theoretically more susceptible to oxidative stress than the other. In this study, we evaluated the susceptibility of each membrane hemileaflet for peroxyl radical-mediated oxidation. In vitro peroxidation of intestinal brush-border membrane was initiated with the peroxyl radical-generator 2,2'-azobis-(2-amidinopropane)hydrochloric acid (AAPH). Oxidation events were monitored by following the oxidation-sensitive degradation of the lipid-soluble fluorescent probe cis-parinaric acid (PnA). The degradation patterns were clearly distinct in the inner and outer hemileaflet. PnA degradation in the inner hemileaflet was consistent with a slow first-order reaction, whereas degradation in the outer leaflet appeared as two first-order processes delayed in time. The results suggest that the sum of available antioxidants and endogenous substrates for oxidation are consumed more rapidly in the outer membrane hemileaflet, making this leaflet more susceptible to peroxidation compared with the cytofacial leaflet.

Ontogenetic development of nutrient transporters in rat intestine

1992 ◽  
Vol 263 (5) ◽  
pp. G593-G604 ◽  
Author(s):  
E. M. Toloza ◽  
J. Diamond
Keyword(s):  
Body Weight Gain ◽  
Safety Margin ◽  
Morphometric Parameters ◽  
Dietary Intakes ◽  
Rat Intestine ◽  
Intestinal Brush Border ◽  
Dry Milk ◽  
Two Peaks

We measured intestinal brush-border uptakes of three sugars and three amino acids, plus intestinal morphometric parameters, in rats from the day of birth until adulthood. Rates of body weight gain had pronounced peaks in the suckling phase and again during weaning, separated by a dip at the onset of weaning. These two peaks coincided with peaks or plateaus in intestinal growth and in glucose (Glc) and proline (Pro) uptake capacities, which may provide the basis for high rates of body growth. Pro uptake declined relative to Glc uptake upon weaning, reflecting decreasing protein needs for growth and decreasing protein intake relative to carbohydrate intake. Fructose (Frc) and lysine uptake increased steeply on weaning, whereas galactose uptake declined relative to that of Glc. Rats prevented from normal weaning by being maintained on dry milk were generally similar to normal rats weaned onto chow. Notably, their Frc uptake still rose steeply on weaning despite low dietary Frc levels, suggesting hard-wired regulation of Frc transporter development. Our in vitro uptakes are similar to modern in vivo values in the same strain of rats. Nutrient uptake capacities exceed normal dietary intakes by only a modest safety margin.

Dietary regulation of intestinal ascorbate uptake in guinea pigs

1991 ◽  
Vol 260 (1) ◽  
pp. G108-G118 ◽  
Author(s):  
W. H. Karasov ◽  
B. W. Darken ◽  
M. C. Bottum
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Brush Border ◽  
Guinea Pigs ◽  
Dietary Regulation ◽  
Intestinal Brush Border ◽  
Pregnancy And Lactation ◽  
Human Adults ◽  
Ascorbate Uptake

We measured ascorbic acid (AA) uptake across the intestinal brush border in vitro in intact tissue from guinea pigs fed maintenance AA (200 mg/kg diet) or made hypervitaminotic (5,000 mg/kg diet) or hypovitaminotic (chronically and acutely). Total uptake per centimeter ileum was 25-50% lower in hypervitaminotic juvenile, adult male, and lactating guinea pigs compared with their respective controls, whereas carrier-mediated D-glucose uptake and Na(+)-independent AA uptake were similar. High dietary ascorbate specifically reduced the Vmax for carrier-mediated AA uptake. Hypovitaminosis had no significant effect on uptake of AA or other solutes. We performed diet-switching experiments (high-AA diet to maintenance diet) with young and adult guinea pigs to determine the reversibility of the downregulation. In adult guinea pigs, the downregulation of AA uptake was reversible within 7 days. In the young of mothers fed high AA during pregnancy and lactation, and which fed on high AA for 14 days after weaning, the downregulation was reversible within 14 days. Thus regulation of AA uptake is reversible and therefore probably does not play a significant role in the development of vitamin C dependency in human adults, or their young, after ingestion of megadoses of ascorbic acid.

In vitro Gastrointestinal Models for Prebiotic Carbohydrates: A Critical Review

2019 ◽  
Vol 25 (32) ◽  
pp. 3478-3483 ◽  
Author(s):  
Oswaldo Hernandez-Hernandez
Keyword(s):  
Small Intestine ◽  
Gastrointestinal Tract ◽  
Brush Border ◽  
Critical Review ◽  
In Vitro Digestibility ◽  
Human Gastrointestinal Tract ◽  
Intestinal Brush Border ◽  
Small Intestinal ◽  
Existing Data

Background: In the last decade, various consortia and companies have created standardized digestion protocols and gastrointestinal simulators, such as the protocol proposed by the INFOGEST Consortium, the simulator SHIME, the simulator simgi®, the TIM, etc. Most of them claim to simulate the entire human gastrointestinal tract. However, few results have been reported on the use of these systems with potential prebiotic carbohydrates. Methods: This critical review addresses the existing data on the analysis of prebiotic carbohydrates by different in vitro gastrointestinal simulators, the lack of parameters that could affect the results, and recommendations for their enhancement. Results: According to the reviewed data, there is a lack of a realistic approximation of the small intestinal conditions, mainly because of the absence of hydrolytic conditions, such as the presence of small intestinal brush border carbohydrases that can affect the digestibility of different carbohydrates, including prebiotics. Conclusion: There is a necessity to standardize and enhance the small intestine simulators to study the in vitro digestibility of carbohydrates.

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