BasicāLiver, Pancreas, and Biliary TractThe human bile salt export pump: Characterization of substrate specificity and identification of inhibitors*,**,*
Section snippets
Generation of full-length ABCB11 complementary DNA
Total RNA from human liver was extracted by using RNAzol B (Biogenesis, Poole, England). First strand complementary DNA (cDNA) synthesis was performed by using C. therm. polymerase (Roche, Basal, Switzerland) in a 20 Ī¼L reaction containing 1 Ć reverse transcriptase buffer, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol, 5% dimethyl sulfoxide, 1 Ī¼mol/L primer, 20 mmol/L deoxynucleoside triphosphates (AB gene, Epsom, England), 10 units RNAase inhibitor (Roche), and 6 units C. therm. polymerase. Products
Cloning of ABCB11 complementary DNA
The ABCB11 coding cDNA of 3.963 kilobases was amplified as 3 overlapping fragments (1, 2, and 3; see Figure 1). Each fragment was amplified with primers to introduce restriction endonuclease sites at the 5' and 3' ends to facilitate their cloning. The 3' end of ABCB11 was modified to encode 6 histidine residues at the carboxyl terminus of the recombinant protein. Unique restriction endonuclease sites in the coding sequence shared between overlapping fragments were used to reconstruct the
Discussion
Normal concentrations of bile salts in post sinusoidal blood are generally <10 Ī¼mol/L. The concentration is kept low by their removal from portal blood by a number of transporters located in the basolateral membrane of hepatocytes.24 The true intrahepatocyte concentration of free bile salts is not known. There are good reasons why concentrations should be kept lowābile salts are highly cytotoxic detergents. This all points to the need for a high-affinity export system for bile salts in the
Acknowledgements
The authors thank Colin Dolphin for advice on insect cell membrane preparations, Charlotte Dyer and Andrew McKnight for their expert molecular biology guidance, David Dewin for critical reading of the manuscript, and Ann Mowat and family for their enthusiastic support.
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Cited by (0)
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Address requests for reprints to: Richard Thompson, M.D., Ph.D., Institute of Liver Studies, King's College Hospital, Denmark Hill, London SE5 9PJ, England. e-mail: [email protected]; fax: (44) 207-346-3700.
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Supported by The Alex Mowat, Ph.D. Studentship Fund (to J.A.B.), The Wellcome Trust (to R.J.T.), The Children's Liver Disease Foundation (to S.S.S.), and The Medical Research Council (to K.J.L. and C.F.H.)
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This article is dedicated to the memory of Professor Alex Mowat, Professor of Pediatric Hepatology at King's College Hospital, London, England (1990ā1995).