Hans-Dieter Riedel

Downregulation of ileal bile acid absorption in bile-duct-ligated rats

BACKGROUND/AIMS Accumulation of toxic bile acids in cholestasis contributes to liver injury and depends on their synthesis, secretion and intestinal absorption. In the present study, we investigated the effect of cholestasis on the active ileal absorption of bile acids in vivo and the adaptation of transporters involved in ileal bile acid absorption. METHODS Male Wistar rats underwent ligation of the common bile duct or biliary diversion. Sham-operated rats served as controls. Active ileal bile acid absorption of taurocholate was measured by an intestinal perfusion technique. Transporter mRNA levels of the Na+/bile acid cotransporting protein (IBAT), ileal lipid binding protein (ILBP) and organic anion transporter subtype 3 (Oatp3) and protein expression of IBAT and ILBP were determined in the distal ileum. RESULTS After bile duct ligation the intestinal absorption rates of taurocholate were lower (p<0.05) and after biliary diversion absorption rates were higher compared to sham-operated animals (p<0.05). The absorption rates were inversely correlated to serum bile acid concentrations. Levels of IBAT-, ILBP- and Oatp3- mRNA were not different between the groups. However, in cholestatic rats, the expression of the 99-kDa dimer of IBAT was decreased compared to controls (p<0.05), whereas the 46-kDa monomeric protein of IBAT and the expression of ILBP was unchanged. After biliary diversion a similar pattern of protein expression was observed, despite an increased absorption rate. CONCLUSIONS Cholestasis leads to a decreased active ileal absorption of taurocholate. The changes in protein expression may not account for the different absorption rates. The intestinal absorption of bile acids seems to be regulated by their systemic concentration.

Hemochromatosis and transferrin receptor gene polymorphisms in chronic hepatitis C: impact on iron status, liver injury and HCV genotype.

Mild iron overload in chronic hepatitis C is associated with liver fibrosis, hepatitis C virus (HCV) genotype 1b infection, and an impaired response to interferon therapy. In this study we evaluated whether polymorphisms in the hemochromatosis gene HFE and the transferrin receptor gene TFR1 are associated with these typical findings. The study considered 246 HCV-infected patients and 200 blood donors as controls, in which C282Y, H63D, and S65C mutations (HFE) and the S142G polymorphism (TFR1) were detected. HCV genotype, serum ferritin levels, stainable intrahepatic iron, and grade of fibrosis according to the METAVIR score (F0–F4) were determined. In HCV-infected patients, heterozygosity for the C282Y mutation in HFE was significantly associated with elevated serum ferritin levels, stainable liver iron, and advanced fibrosis or cirrhosis (F2–F4). By multivariate logistic regression analysis the odds ratio for the development of advanced fibrosis or cirrhosis (F2–F4) was 2.5 for HCV-infected patients carrying a heterozygous C282Y mutation and 4.8 for HCV-infected patients with C282Y/H63D and C282Y/S65C compound heterozygosity. Heterozygosity for the C282Y mutation in HFE contributes to iron accumulation and fibrosis progression in chronic hepatitis C.

S100A9 is a biliary protein marker of disease activity in primary sclerosing cholangitis.

Background and Aims Bile analysis has the potential to serve as a surrogate marker for inflammatory and neoplastic disorders of the biliary epithelium and may provide insight into biliary pathophysiology and possible diagnostic markers. We aimed to identify biliary protein markers of patients with primary sclerosing cholangitis (PSC) by a proteomic approach. Methods Bile duct-derived bile samples were collected from PSC patients (n = 45) or patients with choledocholithiasis (n = 24, the control group). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to analyse the proteins, 2-D-gel patterns were compared by densitometry, and brush cytology specimens were analysed by RT-PCR. Results A reference bile-duct bile proteome was established in the control group without signs of inflammation or maligancy comprising a total of 379 non-redundant biliary proteins; 21% were of unknown function and 24% had been previously described in serum. In PSC patients, the biliary S100A9 expression was elevated 95-fold (p<0.005), serum protein expression was decreased, and pancreatic enzyme expression was unchanged compared to controls. The S100A9 expression was 2-fold higher in PSC patients with high disease activity than in those with low activity (p<0.05). The brush cytology specimens from the PSC patients with high disease activity showed marked inflammatory activity and leukocyte infiltration compared to the patients with low activity, which correlated with S100A9 mRNA expression (p<0.05). Conclusions The bile-duct bile proteome is complex and its analysis might enhance the understanding of cholestatic liver disease. Biliary S100A9 levels may be a useful marker for PSC activity, and its implication in inflammation and carcinogenesis warrants further investigation.

The antirepressor of phage P1 Isolation and interaction with the C1 repressor of P1 and P7

Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in‐frame start codon. Another open reading frame, icd, 5′ upstream of and overlapping ant1 is required for ant1 expression. Using appropriate ant gene‐carrying plasmids we have overproduced and purified Ant½ in the form of a protein complex and Ant2 as a single protein. Sequence analysis confirmed the N‐terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N‐terminal methionine is missing in the Ant2 protein. Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co‐precipitate with the Ant½ complex but not with Ant2 protein alone. The results suggest that the antirepressor may exert its C1‐inactivating function by a direct protein—protein interaction.

Organization of the immunity region immI of bacteriophage P1 and synthesis of the P1 antirepressor.

The immI region of bacteriophage P1 includes the ant/reb gene, which encodes the antirepressor protein, and the c4 gene, which encodes a repressor molecule that negatively regulates antirepressor synthesis. The antirepressor interferes with the activity of the P1 repressor of lytic function, the product of the c1 gene. We have determined the DNA sequences of the immI region of P1 wild-type and the mutants virs, ant16, ant17, and reb22. Using suitable P1 immI DNA subfragments cloned into a vector of the T7 bacteriophage RNA polymerase expression system the antirepressor protein(s) was overproduced. On the basis of positions of immI mutations and the sizes of ant gene products, the following organizational feature of the P1 immI region is suggested: (1) the genes c4 and ant are cotranscribed in that order from the same promoter in the clockwise direction of the P1 genetic map; (2) an open reading frame for an unknown gene is located in between c4 and ant; (3) the site at which the c4 repressor acts is located within the c4 structural gene; (4) two antirepressor proteins of molecular weights 42,000 and 32,000 are encoded by a single open reading frame, with the smaller protein initiating at an in-frame start codon; (5) transcription of immI is regulated via a c1-controlled operator, Op51, indicating a communication between the immunity systems immC and immI.

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase alpha from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase alpha to DNA and conclude that ATP increases the processivity of the enzyme.