R.P.J. Oude Elferink

Assignment of the human UDP glucuronosyltransferase gene (UGT1A1) to chromosome region 2q37

UDP glucuronosyltransferases (UGTs) comprise a multigene family of drug-metabolizing enzymes. The sub-family of UGTs that conjugate bilirubin and phenolic compounds with glucuronic acid has been termed UGT1A1. In man, UGT1A1 isoforms are encoded by a single gene, UGT1A1. Protein isoforms encoded by UGT1A1 originate by alternative splicing. In the present study, we used the cDNA of UGT1A1*4, a bilirubin-conjugating isoform, to localize the UGT1A1 locus in the human genome. The UGT1A1 gene was assigned by in situ hybridization to chromosome region 2q37.

Hepatic, intestinal and renal transport of 1-naphthol-β-D-glucuronide in mutant rats with hereditary-conjugated hyperbilirubinemia

SummaryRecently, a mutant rat strain was described with a genetic defect for the biliary excretion of organic anions (TR− rats). To determine the possible heterogeneity of the transport systems in liver, intestine and kidney we investigated the transport of the anion 1-naphthol-β-d-glucuronide (1-NG) in isolated vascularly perfused organ preparations of the rat liver, intestine and kidney of both Wistar rats and TR− rats. 1-NG was administered as such (liver and kidney experiments) or formed intracellularly from 1-naphthol (1-N) (liver and gut experiments). Independent of the type of exposure to 1-NG, the biliary excretion was considerably impaired in TR− rats. In the intestine the total appearance and the vascular/luminal distribution pattern of 1-NG were not significantly different from the values in control rats. Furthermore, no significant disturbance was found with respect to the renal clearance of 1-NG in the TR− rat when compared with the Wistar rat. Thus, the genetic defect in the TR− rat is restricted to an impaired hepatobiliary excretion of 1-NG and does not affect the excretory systems of the intestine and kidney. These results suggest that the excretion of 1-NG by the liver, intestine and kidney involves distinct organ-specific transport systems.

Characterization of immunoglobulin A kappa autoantibodies to human lactate dehydrogenase isoenzyme-3.

Abstract We have purified with a cumulative recovery of 48% from the serum of a patient the immunoglobulin Aκ subunit of the lactate dehydrogenase-immunoglobulin Aκ (LD-IgAκ) complex. It appears that the pI range of the complex is 5.4–5.8. The Ig part of the complex showed a monoclonal character, and the complex exhibited a 1:2 molar ratio of the Ig to the LD isoenzyme. From reconstitution experiments by two different methods we concluded that LD-3 is essential for restoring the original complex. Additional studies showed a recombination of the IgAκ with both autologous and homologous LD-3, and a binding of LD-3 at the Fab region of the Ig. The estimated value of the affinity constant (Keq) was 2.1 × 109 liters/mol. Analysis of the specific LD-3-binding IgAκ concentrations in the sera of five cases revealed a broad range of the individual immune response. Our first quantitative data on the lymphocyte subpopulations revealed a significantly increased OKT4 OKT8 ratio due to a reduction in the absolute number of T suppressor cells.

Fiber-chimeric adenoviruses expressing fibers from serotype 16 and 50 improve gene transfer to human pancreatic adenocarcinoma

Survival of patients with pancreatic cancer is poor. Adenoviral (Ad) gene therapy employing the commonly used serotype 5 reveals limited transduction efficiency due to the low amount of coxsackie-adenovirus receptor on pancreatic cancer cells. To identify fiber-chimeric adenoviruses with improved gene transfer, a library of Ad vectors based on Ad5 and carrying fiber molecules consisting of 16 other serotypes were transduced to human pancreatic carcinoma cell lines. Adenoviruses containing fibers from serotype 16 and 50 showed increased gene transfer and were further analyzed. In a gene-directed prodrug activation system using cytosine deaminase, these adenoviruses proved to be effective in eradicating primary pancreatic tumor cells. Fiber-chimeric Ad5 containing fiber 16 and wild-type Ad5 were also transduced ex vivo to slices of normal human pancreatic tissue and pancreatic carcinoma tissue obtained during surgery. It was shown that fiber-chimeric Ad5 with fiber 16 revealed an improved gene delivery to primary pancreatic tumor tissue compared to Ad5. In conclusion, fiber-chimeric adenoviruses carrying fiber 16 and 50 reveal a significantly enhanced gene transfer and an increased specificity to human pancreatic adenocarcinoma compared to Ad5, whereas transduction to normal pancreatic tissue was decreased. These findings expand the therapeutic window of Ad gene therapy for pancreatic cancer.

Molecular Targets for the Treatment of Fibrosing Cholangiopathies

Emerging pathophysiologic insights are leading to novel approaches to treating fibrosing cholangiopathies. The current treatment, using ursodeoxycholic acid (UDCA), may slow the progression of some chronic cholangiopathies but cannot heal them. Apart from immunosuppressive interventions aimed at minimizing immune‐mediated damage, the use of specific modifiers of hepatobiliary secretory and cytoprotective mechanisms may eventually give rise to a new class of disease‐modifying anticholangiofibrotic drugs.

Intracellular Transport of Brush-Border and Lysosomal Enzymes in the Human Enterocyte

The mechanisms underlying the biosynthesis, intracellular transport, and sorting of proteins destined for extra- and intracellular use have been extensively studied in polarized epithelial cells during the last few years. Much of our understanding about these mechanisms arose from studies on the intracellular pathways taken by newly synthesized viral membrane glycoproteins in cultured epithelial cells (K. Simons and Fuller 1985, N.L. Simons et al. 1985). However, little information is available about the synthesis and transport of endogenous glycoproteins (Danielsen et al. 1984). To study these processes the intestinal epithelial cell (enterocyte) is an attractive model for a number of reasons.

Regulation of MDR2 P-glycoprotein expression by bile salts

The phosphatidyl translocating activity of the mdr2 P-glycoprotein (Pgp) in the canalicular membrane of the mouse hepatocyte is a rate-controlling step in the biliary secretion of phospholipid. Since bile salts also regulate the secretion of biliary lipids, we investigated the influence of the type of bile salt in the circulation on mdr2 Pgp expression and activity. Male mice were led a purified diet to which either 0.1% (w/w) cholate or 0.5% (w/w) ursodeoxycholate was added. This led to a near-complete replacement of the endogenous bile salt pool (mainly tauromuricholate) by taurocholate or tauroursodeoxycholate respectively. The phospholipid secretion capacity was then determined by infusion of increasing amounts of tauroursodeoxycholate. Cholate feeding resulted in a 55% increase in maximal phospholipid secretion compared with that in mice on the control diet. Northern blotting revealed that cholate feeding increased mdr2 Pgp mRNA levels by 42%. Feeding with ursodeoxycholate did not influence the maximum rate of phospholipid output or the mdr2 mRNA content. Female mice had a higher basal mdr2 Pgp mRNA level than male mice, and this was also correlated with a higher phospholipid secretion capacity. This could be explained by the 4-fold higher basal cholate content in the bile of female compared with male mice. Our results suggest that the type of bile salts in the circulation influences the expression of the mdr2 gene.

694 A BILIARY BICARBONATE UMBRELLA PROTECTS HUMAN CHOLANGIOCYTES AND CHOLANGIOCARCINOMA CELLS AGAINST BILE ACID TOXICITY

Human cholangiocytes are physiologically exposed to millimolar concentrations of glycine-conjugated hydrophobic bile salts, which are toxic to other cell types including hepatocytes already in the micromolar range after cell entry. Apolar, protonated bile acids rather than polar, deprotonated bile salts may passively enter cholangiocytes. pKa and biliary pH determine bile acid protonation status in bile. We hypothesize that biliary HCO−3 secretion in humans serves to maintain an alkaline pH near the apical surface of cholangiocytes and fosters deprotonation of apolar bile acids to polar bile salts. This ‘biliary HCO−3-umbrella’ might be a key protective mechanism of human cholangiocytes against glycineconjugated bile acids (Hepatology 2010; 52: 1489). Our aim was to test if toxicity of unconjugated, glycine-conjugated (pKa 4–5) and taurine-conjugated (pKa < 2) bile salts in cholangiocytes is (i) pH-dependent, and (ii) antagonized by anion exchanger 2 (AE2) activity. Methods: Two human cholangiocarcinomaand one human immortalized cholangiocyte cell line were exposed to glycine-, taurine-, and unconjugated chenodeoxycholate (CDC), from 0.1mM to 2.0mM at pH8.0, 7.4, 7.1, 6.7 and 6.4 and after knockdown of AE2 by shRNA. After four hours, cell viability and apoptosis were determined by WST-1 and caspase-3/-7 assays, respectively. Results: In cholangiocarcinoma cells and non-transformed immortalized cholangiocytes, CDCand GCDC-, but not TCDCinduced cholangiocyte toxicity was pH-dependent and increased gradually when pH was lowered from 7.4 to 7.1, 6.7 or 6.4. E.g., in non-transformed cells, at pH 7.4 no bile salt-toxicity was observed while at pH 6.4 0.5mM and 1mM CDC decreased viability by 80.8±5.3 and 83.5±9.7% (p < 0.01, n = 5) and increased caspase-3/-7 activity 9and 27-fold, respectively (p < 0.001, n = 5). Acidification alone had no effect. AE2 knockdown led to 3-fold and 2-fold apoptosis induced by 0.75mM CDC or 2mM GCDC at pH 7.4 (p < 0.01, n = 6). When increasing pH to 8.0 during bile salt exposure, AE2 knockdown cells were rescued from bile salt toxicity. Conclusion: Our data support the protective function of a biliary HCO−3-umbrella against bile acid-induced cholangiocyte injury. AE2 may serve as a key element of this HCO−3-umbrella. Genetic and acquired functional defects of elements of the biliary HCO−3umbrella may contribute to development and progression of cholangiopathies.

1120 BICARBONATE SECRETION OF MOUSE CHOLANGIOCYTES INVOLVES Na+–HCO3− COTRANSPORT IN ADDITION TO Na+-INDEPENDENT Cl−/HCO3− EXCHANGE

Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.