Adsorption of xenobiotics to plastic tubing incorporated into dynamic in vitro systems used in pharmacological research--limits and progress.

Commonly used materials incorporated into dynamic culture systems typically show the feature of adsorption of lipophilic xenobiotics. Yet, this phenomenon is strongly limiting the use of dynamic culture models and ex vivo organ perfusions in pharmacological and toxicological research. The aim of the study was to characterize different materials with respect to their capacity for drug adsorption and to find methods or materials to reduce the loss of substrate by adsorption in order to improve the use of dynamic in vitro systems. The adsorption of different xenobiotics (lidocaine, midazolam, lormetazepam, phenobarbital, testosterone, ethoxyresoroufine) to tubes used in dynamic in vitro systems (polyvinyl-chloride, silicone) were investigated and compared to a new material (silicone-caoutchouc-mixture). In addition, the role of protein deposition onto the tubing was studied and it was investigated whether it was possible to reach saturation of the inner tube surface by pre-loading it with the test compound. We found that silicone tubes provided the highest comfort with respect to handling and reusability, but they also demonstrated the highest capacity for substrate adsorption. Polyvinyl-chloride was the second best in handling but also demonstrated a high complexity in its adsorption behavior. The silicone-caoutchouc-mixture reached acceptable experimental results with respect to its handling and demonstrated a very low capacity for substrate adsorption.

Comparative analysis of metabolism of medium- and plasma perfused primary pig hepatocytes cultured around a 3-D membrane network.

Culture media are frequently used in the evaluation of metabolical functions of hepatocytes in hybrid liver support systems (hLSS). However, media compositions differ substantially from those of plasma. Therefore, our study was designed to investigate whether current in vitro studies with medium are suitable to assess the metabolical competence of hLSS-cultures during clinical application as well as to explore whether the cell nutrition with medium provides a suitable modus operandi for stand by cultivation. Paired bioreactor cultures were perfused with either Williams' Medium E (MPB) or human plasma (PPB). About 6x108 primary pig hepatocytes (>97% viability) were cultured in three laboratory scale bioreactors designed according to Gerlach's bioreactor-concept. Different perfusion protocols were initiated after a standardised period allowing for cell attachment and reorganisation in aggregates. Whereas patterns of enzyme release were similar in both protocols the metabolical behaviour was different between MPB (anabolic state) and PPB (catabolic state). Furthermore, compared to MPB the lidocaine-MEGX-tests for PPB demonstrated lower MEGX-concentrations and a different reaction pattern. We conclude that the nutrition of hepatocytes with medium during the stand by period itself might influence the cell function and subsequently the efficacy of the hLSS-treatment during clinical application.

The phosphate carrier from yeast mitochondria. Dimerization is a prerequisite for function.

Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and recombinant PIC proteins with different C-terminal extensions were expressed in Escherichia coli as inclusion bodies. From these, PIC was isolated with the detergent sodium lauroyl sarcosinate in a form, partially monomeric and unfolded. This PIC associates to stable dimers after exchanging the detergent to the polyoxyethylene detergent C12E8 and dialysis. Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo- and heterodimeric forms of PIC, which are all fully active after reconstitution. As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer. We investigated its dimeric nature in the functionally active state after reconstitution. When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linear dependence. Heterodimeric PIC constructs consisting of both an active and an inactivated subunit do not catalyze phosphate transport. In contrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier. These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, and (iii) that transport catalyzed by PIC dimers involves functional cross-talk between the two monomers.

Mutations of the DPC4/Smad4 gene in biliary tract carcinoma.

A candidate tumor suppressor gene, DPC4, located at 18q21.1, has recently been shown to be inactivated in half of pancreatic adenocarcinomas. The close developmental relationship of the pancreas and biliary tract prompted us to determine the role of DPC4 in the multistep carcinogenesis of biliary tract carcinoma. A search for mutations in the genomic sequence of the highly conserved COOH-terminal domain of DPC4 (exons 8-11) was performed by single-strand conformational polymorphism analysis. Five of 32 (16%) primary biliary tract carcinomas had point mutations in the DPC4 sequence. Interestingly, inactivation of DPC4 was especially common in carcinomas originating from the common bile duct (four of eight specimens analyzed), suggesting an important role for DPC4 in the development of this subtype of biliary tract tumor.

The reversible antiport-uniport conversion of the phosphate carrier from yeast mitochondria depends on the presence of a single cysteine.

Wild type and mutant phosphate carriers (PIC) from Saccharomyces cerevisiae mitochondria were expressed in Escherichia coli as inclusion bodies, solubilized, purified, and optimally reconstituted into liposomal membranes. This PIC can function as coupled antiport (Pi-/Pi- antiport and Pi- net transport, i.e. Pi-/OH- antiport) and uncoupled uniport (mercuric chloride-induced Pi- efflux). The basic kinetic properties of these three transport modes were analyzed. The kinetic properties closely resemble those of the reconstituted PIC from beef heart mitochondria. A competitive inhibitor of phosphate transport by the PIC, phosphonoformic acid, was used to establish functional overlap between the the physiological transport modes and the induced efflux mode. Replacement mutants were used to relate the reversible switch from antiport to uniport to a specific residue of the carrier. There are only three cysteines in the yeast PIC. They are at positions 28, 134, and 300 and were replaced by serine, both individually and in combinations. Cysteine 300 near the C-terminal loop and cysteine 134 located within the third transmembrane segment are accessible to bulky hydrophilic reagents from the cytosolic side, whereas cysteine 28 within the first transmembrane segment is not. None of the three cysteines is relevant to the two antiport modes. Cysteine 134 was identified to be the major target of bulky SH reagents, that lead to complete inactivation of the physiological transport modes. The reversible conversion between coupled antiport and uncoupled uniport of the PIC depends on the presence of one single cysteine (cysteine 28) in the PIC monomer, i.e. two cysteines in the functionally active dimer. The consequences of this result with respect to a functional model of the carrier protein are discussed.

Bacterial overexpression of putative yeast mitochondrial transport proteins.

Thirty-two genes have been identified within the genome of the yeast Saccharomyces cerevisiae which putatively encode mitochondrial transport proteins. We have attempted to overexpress a subset of these genes, namely those which encode mitochondrial transporters of unknown function, and have succeeded in overexpressing 19 of these genes. The overexpressed proteins were then isolated and tested for five well-characterized reconstituted transport activities (i.e., the transport of citrate, dicarboxylates, pyruvate, camitine, and aspartate). Utilizing this approach, we have clearly identified the yeast mitochondrial dicarboxylate transport protein, as well as two additional lower-magnitude transport functions (i.e., tricarboxylate and dicarboxylate transport activities). The implications of these results and the considerations relevant to this approach are discussed.