Evaluation of coupling of cobalamin to antisense oligonucleotides by thin-layer and reversed-phase liquid chromatography

Guy, Muriel; Olszewski, A.; Monhoven, Nathalie; Namour, Farès; Guéant, Jean‐Louis; Plénat, François

doi:10.1016/s0378-4347(97)00534-3

Cited by 6 publications

(1 citation statement)

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“…The conjugate was prepared as previously described [13]. The avidin/biotinyl-Cbl complex was purified through a Superose 12 column and identified by UV-visible spectrophotometer at 360 nm.…”

Section: Preparation and Identification Of Avidin/biotinyl-cobalamin mentioning

confidence: 99%

Paracellular Transport of Avidin Saturated or not with Biotinylated Cobalamin through Caco-2 Cell Epithelium Monolayer

Meyer

Pons

Namour

et al. 2001

Cell Physiol Biochem

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Background: The cationic charge of molecules may promote their uptake across epithelia, which are rich in brush border anionic sites. The transport of unsaturated avidin and avidin saturated with a biotinylated compound was investigated across Caco-2 adenocarcinoma cell with fetal enterocyte phenotype. Methods: The unsaturated avidin and avidin saturated with either biotin or a biotinyl-cobalamin conjugate (biotinyl-Cbl) were iodinated to follow their transport through the cell monolayer. Their apparent permeability coefficient (Papp) and transepithelial pathway were determined and compared to those for control radiolabeled markers [³H]-mannitol, [¹²⁵I]-β-lactoglobulin and [⁵⁷Co]-cobalamin/intrinsic factor (Cbl/IF). Results: The Papp of [¹²⁵I]-avidin estimated at 2.8 x 10^-7 ± 0.08 cm/ s was close to that for mannitol that uses paracellular pathway. The binding of biotin or biotin conjugate to avidin enhanced its tetrameric conformation. The Papp for [¹²⁵I]-avidin/biotin and [¹²⁵I]- avidin/biotinyl-Cbl were respectively increased by 2-fold, compared to that for [¹²⁵I]-avidin and 4-fold, compared to that for [¹²⁵I]-β-lactoglobulin and [⁵⁴Co]-Cbl/IF. The protein was not accumulated in the cell and was found in intact form in the basolateral side, after its transport across the monolayer. Chloroquine (0.66 µmol/ml) did not significantly decrease the Papp for [¹²⁵I]-avidin/biotinyl-Cbl. Conversely it decreased by 80% the Papp for Cbl/IF, that uses transepithelial pathway. Conclusions: Avidin (either saturated or not with biotin and biotinyl-Cbl) was able to cross the monolayer of Caco-2 cell line through a paracellular pathway. This study pointed out the interest for using this protein as a shuttle for increasing the transport rate of biotinylated compounds through fetal epithelial barriers.