Expression of a bile acid transporter in biliary epithelial cells from normal and cholestatic rat livers.

A sodium dependent bile acid carrier has recently been cloned and characterized in rat ileum. The present study demonstrates the presence of a mRNA species specific for the rat ileal bile acid carrier (r-IBAT) in rat biliary epithelial cells. Moreover, immunohistochemistry with a peptide specific antibody demonstrates protein expression in biliary epithelial cells from normal and bile duct ligated rat livers. Besides a cytoplasmic staining a predominant staining of the apical membrane could be observed. These observations indicate that biliary epithelial cells are involved in bile acid transport across the biliary tree. In addition the carrier could also play a role in the signal transduction of bile acid induced ductular secretion.

Chlorambucil-taurocholate is transported by bile acid carriers expressed in human hepatocellular carcinomas.

BACKGROUND & AIMS: Chemotherapy of hepatocellular carcinomas is hampered by the insufficient accumulation of cytostatic drugs within the tumor cells. The aim of this study was to evaluate the feasibility of therapeutic strategies using antineoplastic agents coupled to bile acids. METHODS: Expression of the Na(+)-taurocholate-cotransporting polypeptide (NTCP) was analyzed in six hepatocellular carcinomas and in nonmalignant liver tissue. Uptake of the cytostatic drug [3H]-chlorambucil-taurocholate (S2676) was measured in Xenopus laevis oocytes injected with total messenger RNA (mRNA) from the carcinomas or peritumor tissue or with complementary RNA encoding the NTCP or the organic anion-transporting polypeptide (OATP) of human liver. RESULTS: Expression of hepatocellular carcinoma mRNA in oocytes resulted in mainly Na(+)-dependent uptake of chlorambucil-taurocholate. The level of NTCP mRNA in carcinomas amounted to 56% +/- 27% compared with peritumor tissue. Immunofluorescence studies confirmed the expression of NTCP on the surface of hepatocellular carcinoma cells. OATP expression, determined by immunoblotting, was similar in hepatocellular carcinomas and surrounding liver tissue (n = 3). NTCP mediated Na(+)-dependent uptake of chlorambucil-taurocholate (Michaelis constant, 11 mumol/L), whereas OATP mediated Na(+)-independent uptake. CONCLUSIONS: Hepatocellular carcinomas express the Na(+)-dependent bile acid transporter NTCP. Because NTCP mediates high-affinity uptake of chlorambucil-taurocholate, targeting of cytostatic bile acids to hepatocellular carcinomas could become a feasible therapeutic strategy.

Serine phosphorylation site of the 46-kDa mannose 6-phosphate receptor is required for transport to the plasma membrane in Madin-Darby canine kidney and mouse fibroblast cells.

Up to 4% of the human 46-kDa mannose 6-phosphate receptor (MPR46) expressed in Madin-Darby canine kidney (MDCK) cells are localized at the cell surface. At steady state, the expression of MPR46 on the apical surface of filter-grown MDCK cells is about sixfold lower than on the basolateral surface. The cytoplasmic domain of the MPR46 is phosphorylated on serine 56 at low stoichiometry. By expressing mutant MPR46 we have shown that the MPR46 phosphorylation site is required for delivery to the plasma membrane. In addition, mutant MPR46 expressed in MPR-deficient mouse embryonic fibroblasts were not detected at the cell surface and their ability to sort newly synthesized cathepsin D was not altered. Since the loss of MPR46 phosphorylation correlates with the lack of cell surface expression, phosphorylation of serine 56 may either function as a direct plasma membrane targeting signal or inhibit MPR46 recycling from endosomes to Golgi, resulting in trafficking to the cell surface.

The carboxy-terminal peptides of 46 kDa and 300 kDa mannose 6-phosphate receptors share partial sequence homology and contain information for sorting in the early endosomal pathway.

Recycling of mannose 6-phosphate receptors was investigated by microinjection of F(ab) fragments against their carboxy-terminal peptides (residues 54-67 or 150-164 of the cytoplasmic domain of 46 kDa and 300 kDa mannose 6-phosphate receptor, respectively). For each receptor, masking the carboxy-terminal peptide by the corresponding F(ab) fragments resulted in complete depletion of the intracellular pool. Redistributed 300 kDa mannose 6-phosphate receptor was shown to accumulate at the plasma membrane and to internalize anti-ectodomain antibodies. Internalization of anti-ectodomain antibodies was also observed for redistributed 46 kDa mannose 6-phosphate receptor. Semiquantitative analysis suggested that for both redistributed receptors the amount of intracellularly accumulated anti-ectodomain antibodies was reduced. In addition, downstream transport along the endosomal pathway was slowed down. These data suggest that sorting information for early steps in the endocytic pathway is contained within the carboxy-terminal peptides of mannose 6-phosphate receptors.

Tail-specific antibodies that block return of 46,000 M(r) mannose 6-phosphate receptor to the trans-Golgi network.

Recycling of 46,000 M(r) mannose 6-phosphate receptor (MPR 46) was investigated by microinjection of Fab fragments against small epitopes within the cytoplasmic domain of the receptor. Fab fragments against the peptide 43-47 (Ala-Tyr-Arg-Gly-Val) efficiently blocked return of MPR 46 to the TGN. Antibody-induced redistribution resulted in accumulation of MPR 46 within an endosomal compartment, from which it recycled to the plasma membrane. Rab5 and rab7, markers for early and late endosomes, respectively, were not detectable in the compartment of redistributed MPR 46, suggesting that it represents a specialized endosomal subcompartment. The bulk of redistributed MPR 46 did not colocalize with endocytosed fluid-phase marker, suggesting that it accumulates at a site where MPR 46 has been segregated from endocytosed material, which is destined for transport to lysosomes. Peptide 43-47 contains a tyrosine (residue 44) which has been shown earlier to be part of an internalization signal for MPR 46 (Johnson, K. F., W. Chan, and S. Kornfeld. 1990. Proc. Natl. Acad. Sci. USA. 87:10010-10014). The role of tyrosine residue 44 as part of a putative multifunctional sorting signal is discussed.

Bedside monitoring of anti-coagulation under LDL apheresis by means of aPTT testing with a coagulation monitor.

Optimally adjusted anticoagulation under LDL apheresis is essential for successful treatment: Excessive anticoagulation exposes the outpatient to the risk of uncontrolled hemorrhage, insufficient anticoagulation may shorten the duration of utilization of the immune-adsorption columns. A new device (coagulation monitor 512, Ciba Corning) allows individual adaptation of dosage and timing of heparin application by modifying the standard schedule (5,000 IU intravenously before treatment, 2,000 IU/h continuously). APTT was measured before and after application of heparin and then at 30-min intervals both by the coagulation monitor and by conventional laboratory methods; the respective heparin concentration was determined in addition. The correlation coefficient between the heparin concentration and both the monitor-derived and the laboratory-derived aPTT was 0.811 and 0.590, respectively. An explanation for this finding might be that the monitor avoids the influences induced by subsequent collection and testing of samples associated with laboratory procedures.

Cannibalism and coprophagy are modes of transmission of Blastocrithidia triatomae (Trypanosomatidae) between triatomines.

Transovarial transmission was not detectable among Blastocrithidia triatomae-infected Triatoma infestans. Rather, B. triatomae was transmitted directly between triatomines by cannibalism and coprophagy. Cannibalism conditions that excluded coprophagy always resulted in an infection of Dipetalogaster maxima. The efficiency of transmission was not influenced by the blood source--mice or chickens--fed to the infected donor bugs although chicken blood lyses the epimastigotes of the stomach population. Triatoma infestans was infected by coprophagy only if fed, not if unfed. Blastocrithidia triatomae in dry feces was taken up only if the feces were redissolved in fresh feces. Infections also appeared in groups of bugs fed on chickens previously used for feeding infected bugs.

Colonization of the rectum of Triatoma infestans by Trypanosoma cruzi studied by scanning electron microscopy: influence of blood uptake by the bug.

The colonization of the different regions of the rectum of Triatoma infestans by Trypanosoma cruzi was studied in unfed larvae, during and after feeding of the bug by scanning electron microscopy. The rectal pads always possessed the highest population densities, but in some bugs the main rectal sac and the region around the anus were also covered by a "carpet" of flagellates. Such high densities were never observed at the midgut/rectal junction. A slight decrease in this region might be caused by blood ingestion and the resulting excretion of urine. However, the flagellates in this region cannot be responsible for a phenomenon described by other authors, namely that the percentage of metacyclics is low in the first drop of faeces and increases in the following drops of deposited urine. Our observations indicate that metacyclics lying on the "carpet" of flagellates in bugs before and right after the start of feeding might later become loosened. In all dissections, numerous metacyclics were attached to the rectal wall. Further studies are necessary to clarify the exact origin of metacyclics in the urine.

Colonization of the rectum of Triatoma infestans by Trypanosoma cruzi: influence of starvation studied by scanning electron microscopy.

The colonization of the different regions of the rectum of Triatoma infestans by a Trypanosoma cruzi strain (zymodeme I) originating from the same locality as the bugs was studied by scanning electron microscopy after different periods of starvation of the bugs. Throughout the first 16 weeks no changes in colonization pattern could be observed. Parasite density was always minimal at the midgut/rectal junction and highest on the rectal pads; it was at a similar level in the other three regions of the rectum. Twenty weeks after feeding, a proportion of the bugs had died and in the surviving larvae a decreasing colonization of the cuticle occurred. Nonetheless, despite other regions being flagellate-free, a residual T. cruzi population always remained attached to the rectal pads. No changes in the proportion of trypomastigotes to epimastigotes were observed as starvation progressed.

Scanning electron microscopic studies of Trypanosoma cruzi in the rectum of its vector Triatoma infestans.

The cuticular surface of the rectum of Triatoma infestans and its colonization by a Trypanosoma cruzi strain originating from the same locality as the bugs were studied by scanning electron microscopy at different weeks post infectionem. On the basis of the cuticular folding, the rectum can be subdivided into five regions. The rectal gland has the finest structure and the region anterior to the anus the deepest folds. Try. cruzi always prefers to colonize the rectal gland, while the other regions are colonized in varying densities. Most of the flagellates are epimastigotes (long and short), except in the region at the entrance of the midgut, where trypomastigotes predominate.