Mgl Elferink

Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy‐coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton‐ and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.

Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity.

The microarray technology, developed for the simultaneous analysis of a large number of genes, may be useful for the detection of toxicity in an early stage of the development of new drugs. The effect of different hepatotoxins was analyzed at the gene expression level in the rat liver both in vivo and in vitro. As in vitro model system the precision-cut liver slice model was used, in which all liver cell types are present in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process involving not only hepatocytes but also other cell types such as Kupffer and stellate cells. As model toxic compounds lipopolysaccharide (LPS, inducing inflammation), paracetamol (necrosis), carbon tetrachloride (CCl(4), fibrosis and necrosis) and gliotoxin (apoptosis) were used. The aim of this study was to validate the rat liver slice system as in vitro model system for drug-induced toxicity studies. The results of the microarray studies show that the in vitro profiles of gene expression cluster per compound and incubation time, and when analyzed in a commercial gene expression database, can predict the toxicity and pathology observed in vivo. Each toxic compound induces a specific pattern of gene expression changes. In addition, some common genes were up- or down-regulated with all toxic compounds. These data show that the rat liver slice system can be an appropriate tool for the prediction of multi-cellular liver toxicity. The same experiments and analyses are currently performed for the prediction of human specific toxicity using human liver slices.

Coordinated induction of drug transporters and phase I and II metabolism in human liver slices.

Although regulation of phase I drug metabolism in human liver is relatively well studied, the regulation of phase II enzymes and of drug transporters is incompletely characterized. Therefore, we used human liver slices to investigate the PXR, CAR and AhR-mediated induction of drug transporters and phase I and II metabolic enzymes. Precision-cut human liver slices were incubated for 5 or 24h with prototypical inducers: phenobarbital (PB) (50 microM) for CAR, beta-naphthoflavone (BNF) (25 microM) for AhR, and rifampicin (RIF) (10 microM) for PXR, and gene expression of the phase I enzymes CYP1A1, 1A2, 3A4, 3A5, 2B6, 2A6, the phase II enzymes UGT1A1 and 1A6, and the transporters MRP2, MDR1, BSEP, NTCP and OATP8 was measured. BNF induced CYP1A1, UGT1A1 and UGT1A6 and MRP2, NTCP and MDR1. RIF induced CYP3A4, 3A5, 2B6, 2A6, UGT1A1, UGT1A6 and BSEP, MRP2 and MDR1 and slightly downregulated OATP8. PB induced CYP3A4, 3A5, 2B6 and 2A6, UGT1A1 and all transporters. Large interindividual differences were found with respect to the level of induction. Enzyme activity of CYP3A4, measured by testosterone metabolism, was increased after 24h by RIF. 7-Ethoxycoumarin O-deethylation activity, mediated predominantly by CYP 1A1/1A2 but also by other CYPs, was increased after 24h with PB. We have shown that regulation of all phases of the (in)activation of a drug via the CAR, AhR and the PXR pathways can be studied in human liver slices. The concomitant induction of metabolic enzymes and transporters shows that also in the human liver transporters and metabolic enzymes are regulated coordinately.

Gene expression analysis of precision-cut human liver slices indicates stable expression of ADME-Tox related genes☆

In the process of drug development it is of high importance to test the safety of new drugs with predictive value for human toxicity. A promising approach of toxicity testing is based on shifts in gene expression profiling of the liver. Toxicity screening based on animal liver cells cannot be directly extrapolated to humans due to species differences. The aim of this study was to evaluate precision-cut human liver slices as in vitro method for the prediction of human specific toxicity by toxicogenomics. The liver slices contain all cell types of the liver in their natural architecture. This is important since drug-induced toxicity often is a multi-cellular process. Previously we showed that toxicogenomic analysis of rat liver slices is highly predictive for rat in vivo toxicity. In this study we investigated the levels of gene expression during incubation up to 24 h with Affymetrix microarray technology. The analysis was focused on a broad spectrum of genes related to stress and toxicity, and on genes encoding for phase-I, -II and -III metabolizing enzymes and transporters. Observed changes in gene expression were associated with cytoskeleton remodeling, extracellular matrix and cell adhesion, but for the ADME-Tox related genes only minor changes were observed. PCA analysis showed that changes in gene expression were not associated with age, sex or source of the human livers. Slices treated with acetaminophen showed patterns of gene expression related to its toxicity. These results indicate that precision-cut human liver slices are relatively stable during 24h of incubation and represent a valuable model for human in vitro hepatotoxicity testing despite the human inter-individual variability.

An ex vivo human model system to evaluate specificity of replicating and non-replicating gene therapy agents

Inefficiency, aspecificity and toxicity of gene transfer vectors hamper gene therapy from showing its full potential. On this basis significant research currently focuses on developing vectors with improved infection and/or expression profiles. Screening assays with validity to the clinical context to determine improved characteristics of such agents are not readily available since this requires a close relationship to the human situation. We present a clinically relevant tissue slice technology to preclinically test improved vector characteristics.

Energy transduction and transport processes in thermophilic bacteria

Bacterial growth at the extremes of temperature has remained a fascinating aspect in the study of membrane function and structure. The stability of the integral membrane proteins of thermophiles make them particularly amenable to study. Respiratory enzymes of thermophiles appear to be functionally similar to the mesophilic enzymes but differ in their thermostability and unusual high turnover rates. Energy coupling at extreme temperatures seems inefficient as suggested by the high maintenance coefficients and the high permeability of the cell membrane to protons. Nevertheless, membranes maintain their structure at these extremes through changes in fatty acid acyl chain composition. Archaebacteria synthesize novel membrane-spanning lipids with unique physical characteristics. Thermophiles have adapted to life at extreme temperatures by using sodium ions rather than protons as coupling ions in solute transport. Genetic and biochemical studies of these systems now reveal fundamental principles of such adaptations. The recent development of reconstitution techniques using membrane-spanning lipids allows a rigorous biochemical characterization of membrane proteins of extreme thermophiles in their natural environment.

Slow fusion of liposomes composed of membrane-spanning lipids

The fusion characteristics of large unilamellar liposomes composed of bipolar tetraether lipids extracted from the thermophilic archaeon Sulfolobus acidocaldarius, was investigated. These lipids span the entire membrane and form single monolayer liposomes in aqueous media [Elferink, M.G.L., de Wit, J.G., Demel, R., Driessen, A.J.M. and Konings, W.N., (1992) J. Biol. Chem. 267, 1375-1381]. In the presence of calcium-phosphate, slow mixing of the aqueous liposome contents and membrane lipids occurred, demonstrating that these liposomes are fusion-competent. The fusion process was essentially nonleaky. The rate of fusion increased with the pH and the concentration of calcium and phosphate. Fusion resulted in an increase of the size of the liposomes. These data demonstrate that a monolayer organization of lipids in a membrane does not per se interfere with membrane fusion competence.

THE INTERACTION BETWEEN ELECTRON-TRANSFER, PROTON MOTIVE FORCE AND SOLUTE TRANSPORT IN BACTERIA

The properties of proton solute symport have been studied inStreptococcus cremoris, Rhodopseudomonas sphaeroides andEscherichia coli. In the homolactic fermentative organismS. cremoris the efflux of lactate is a membrane proteinmediated process, which can lead to the generation of a proton motive force. These observations support the energy-recycling model that postulates the generation of metabolic energy by end-product efflux. Studies with oxidants and reductants and specific dithiol reagents inE. coli membrane vesicles demonstrated the presence of two redox-sensitive dithiol-disulphide groups in the transport proteins of proline and lactose. The redox state of these groups is controlled by the redox potential of the environment and by the proton motive force. One redox-sensitive group is located at the inner surface, the other at the outer surface of the membrane. InRps. sphaeroides andE. coli the activity of several transport proteins depends on the activity of the electron transfer systems.On the basis of these results a redox model for proton solute transport coupled in parallel to the electron transfer system is postulated.

Solute Transport Across Bacterial Membranes

The cell-envelope of bacteria is composed of a cytoplasmic membrane which is surrounded by a cellwall, murien- or peptidoglycan layer, and outside the cellwall in Gram-negative bacteria by an outer membrane (lipopolysaccharide layer). Both the outer membrane and the cellwall are freely permeable for small solutes and do not form an osmotic barrier of the cell. This function is exclusively fulfilled by the cytoplasmic membrane.

DIRECT INTERACTIONS BETWEEN ELECTRON-TRANSFER CHAINS AND SOLUTE TRANSPORT-SYSTEMS IN RHODOPSEUDOMONAS-SPHAEROIDES AND ESCHERICHIA-COLI

The intracellular calcium concentration in bacteria is kept well below that of the surrounding medium as long as metabolic energy is available. A system capable of extruding calcium against its concentration gradient is therefore needed. Calcium/proton antiporters, which couple calcium extrusion to the ApH component of the proton motive force have been identified in a number of gram-positive and gram-negative bacteria. Since the natural environment of Streptococcus cremoris (milk) contains a high concentration of calcium, it is very likely that similar mechanisms exist in this organism. An artificial pH gradient (interior acid) across the membrane of K+-loaded membrane vesicles of S. cremoris can be established by nigericin-catalysed K +-efflux. This pH-gradient drives the uptake of calcium. In the absence of a pH-gradient no uptake of calcium is observed. The uncoupler CCCP completely inhibits uptake of calcium. In order to study in more detail the role of the proton motive force, e.g. ApH and At) in calcium extrusion, the light-driven proton pump bacteriorhodopsin was incorporated into the S. cremoris membrane. Membrane vesicles of S. cremoris can be fused with bacteriorhodopsin proteoliposomes by treatment at tow pH (pH ~ 5.5). This fusion process is strictly dependent on the presence of negatively charged phospholipids in the liposomal membrane. Analysis of the fused membranes on a sucrose gradient reveals a peak with a density intermediate of the membrane vesicles and the proteoliposomes. The density of this peak decreases with increasing cardiolipin content of the proteoliposomes. The orientation of bacteriorhodopsin in the fused membranes is inside-out with respect to the in vivo orientation in Halobacterium halobium. In the light a A~, interior positive, is generated. The proton motive force generated by bacteriorhodopsin upon illumination can drive calcium uptake in the fused membranes. Addition of the ionophore valinomycin stimulates calcium uptake and leads to an increase of the ApH. No or a low level of calcium uptake is observed in the dark, in the presence of nigericin, which dissipates the ApH, or in the presence of the uncoupler CCCP. Bleaching of bacteriorhodopsin in the presence of hydroxylamine also abolishes calcium uptake. These findings indicate that a calcium/proton antiport mechanism is operational in S. cremoris.