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1.
Drug Metab Pharmacokinet ; 26(2): 145-53, 2011.
Article in English | MEDLINE | ID: mdl-21127384

ABSTRACT

Caco-2 and T84 cells are intestinal epithelial model cells. Caco-2 cells are more commonly used in drug transport studies, whereas only a few studies have used T84 cells, and the two cell lines have not been compared. We cultured Caco-2 and T84 cells on plastic dishes or polycarbonate Transwell filters and compared the expression and function of ATP binding cassette (ABC) transporters, including multidrug resistance protein (MDR) 1 and multidrug resistance-associated protein (MRP) 2 and MRP3, in response to various compounds. Overall, the pattern of change in transporter mRNA expression in response to compounds was very similar regardless of culture conditions (plastic dish or polycarbonate filter) and cell line (Caco-2 or T84), and changes in MDR1 function was accompanied by expression changes. The cells cultured on Transwell filters were more sensitive to the tested compounds, regardless of the cell line. On comparing the two cell lines, the intrinsic function of MDR1 was stronger in Caco-2 cells, while sensitivity to the tested compounds was more prominent in T84 cells. These results suggest that Caco-2 cells are more suitable for identifying whether MDR1 mediates drug transport, while T84 cells are more useful for assessing the induction capacity of compounds.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Multidrug Resistance-Associated Proteins/biosynthesis , ATP Binding Cassette Transporter, Subfamily B , Bilirubin/pharmacology , Biological Transport, Active/drug effects , Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Cholates/pharmacology , Epithelial Cells/drug effects , Humans , Multidrug Resistance-Associated Protein 2 , RNA/analysis , Taurocholic Acid/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Xenobiotics/pharmacology
2.
Biochem Biophys Res Commun ; 392(3): 421-5, 2010 Feb 12.
Article in English | MEDLINE | ID: mdl-20079714

ABSTRACT

Leukotriene C(4) (LTC(4)) is synthesized by binding of glutathione to LTA(4), an epoxide derived from arachidonic acid, and further metabolized to LTD(4) and LTE(4). We previously prepared a monoclonal antibody with a high affinity and specificity to LTC(4). To explore the structure of the antigen-binding site of a monoclonal antibody against LTC(4) (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC(4) comparable to mAbLTC. The scFvLTC also bound to LTD(4) and LTE(4) with 48% and 17% reactivities, respectively, as compared with LTC(4) binding, whereas the antibody showed almost no affinity for LTB(4).


Subject(s)
Antibodies, Monoclonal/biosynthesis , Immunoglobulin Light Chains/biosynthesis , Leukotriene C4/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Cloning, Molecular , DNA, Complementary/genetics , Immunoglobulin Light Chains/genetics , Mice , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
3.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 64(Pt 11): 1027-30, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18997333

ABSTRACT

Prostaglandin E(2) is a major lipid mediator that regulates diverse biological processes. To elucidate how prostaglandin E(2) is recognized specifically by its antibody, the Fab fragment of a monoclonal anti-prostaglandin E(2) antibody was prepared and its complex with prostaglandin E(2) was crystallized. The stable Fab-prostaglandin E(2) complex was prepared by gel-filtration chromatography. Crystals were obtained by the microbatch method at 277 K using polyethylene glycol 4000 as a precipitant. A diffraction data set was collected to 2.2 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.3, b = 81.8, c = 82.2 A. The asymmetric unit was suggested to contain one molecule of the Fab-prostaglandin E(2) complex, with a corresponding crystal volume per protein weight of 2.75 A(3) Da(-1).


Subject(s)
Antibodies, Monoclonal/chemistry , Antigen-Antibody Complex/chemistry , Dinoprostone , Immunoglobulin Fab Fragments/chemistry , Animals , Antibodies, Monoclonal/immunology , Crystallization , Crystallography, X-Ray , Dinoprostone/chemistry , Dinoprostone/immunology , Immunoglobulin Fab Fragments/immunology , Ligands , Mice , Molecular Structure , X-Ray Diffraction
4.
Artif Organs ; 27(7): 598-604, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12823414

ABSTRACT

The purpose of this study is to utilize the thermo-reversible gelation polymer in which the sol-gel transitting phase is reversibly changed by temperature in a three-dimensional culture system. Human cancer cells have been observed to form multicellular spheroids, whereas fibroblasts slowly develop into small spheroids with the culture medium including this polymer. This polymer has some advantages for use as a culture material, as follows: first, cancer cells grow three-dimensionally in the aqueous solution of this polymer; second, it is easy to harvest cells or spheroids in the aqueous solution of this polymer by simply cooling down the temperature; and third, the culture medium including this polymer is so translucent that the cells or spheroids can be observed through a phase-contrast microscope. We thus conclude that this polymer is a very useful material for three-dimensional cultures.


Subject(s)
Cell Line, Tumor/cytology , Culture Media , Polymers , Spheroids, Cellular/cytology , Cell Culture Techniques/methods , Cell Division , Gels , Humans , Temperature
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