Marieke G. L. Elferink

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein‐dependent ABC transporters

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80°C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high‐affinity binding protein‐dependent ATP‐binding cassette (ABC) transporter. Sugar‐binding studies revealed the presence of four additional membrane‐bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic‐binding protein that is processed at an unusual site at its amino‐terminus; (ii) di‐ and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein‐dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.

Stability and proton-permeability of liposomes composed of archaeal tetraether lipids

Liposomes composed of tetraether lipids originating from the thermoacidophilic archaeon Sulfolobus acidocaldarius were analyzed for their stability and proton permeability from 20 degrees C up to 80 degrees C. At room temperature, these liposomes are considerably more stable and have a much lower proton permeability than liposomes composed of diester lipids originating from the mesophilic bacterium Escherichia coli or the thermophilic bacterium Bacillus stearothermophilus. With increasing temperature, the stability decreased and the proton permeability increased for all liposomes. Liposomes composed from tetraether lipids, however, remain the most stable. These data suggest these liposomes retain the rigidity of the cytoplasmic membrane of S. acidocaldarius needed to endure extreme environmental growth conditions.

Cellobiose uptake in the hyperthermophilic archaeon Pyrococcus furiosus is mediated by an inducible, high-affinity ABC transporter

The hyperthermophilic archaeon Pyrococcus furiosus can utilize different beta-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K(m) = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosus membranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtA gene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.

Analysis of bile acid-induced regulation of FXR target genes in human liver slices

Information about the role of nuclear receptors has rapidly increased over the last decade. However, details about their role in human are lacking. Owing to species differences, a powerful human in vitro system is needed. This study uses for the first time precision‐cut human liver slices in the nuclear receptor field. The farnesoid X receptor (FXR) was chosen as a model. We were able to demonstrate that human liver slices efficiently take up bile acids and show a stable expression of a wide variety of genes relevant for bile acid metabolism, including bile acid transporters, cytochrome P450 enzymes and transcription factors. Treatment with chenodeoxycholate induced small heterodimer partner, bile salt export pump and p‐glycoprotein, ABCB4 and repressed cholesterol 7α hydroxylase, hepatocyte nuclear factor (HNF)1, HNF4 and organic anion transporting peptide (OATP)1B1. OATP1B3, FXR, HNF3β and cytochrome P450 enzyme remained relatively constant. In contrast to what has been observed in mice and rat studies, SHP induction did not result in repression of sodium‐dependent bile acid cotransporter expression. Further, regulation of genes seemed to be dependent on concentration and time. Taken together, the study shows that the use of liver slices is a powerful technique that enables to study nuclear receptors in the human liver.

IMMUNOCHEMICAL ANALYSIS OF MEMBRANE-VESICLES AND CHROMATOPHORES OF RHODOPSEUDOMONAS-SPHAEROIDES BY CROSSED IMMUNOELECTROPHORESIS

Two types of vesicular membranes can be isolated from phototrophically grown Rhodopseudomonas sphaeroides [l]. So-called membrane vesicles are derived from the cytoplasmic membrane by osmotic lysis of lysozyme-EDTA-treated cells. Alternatively chromatophores can be derived from invaginations of the cytoplasmic membrane by French press disruption of intact cells [ 11. Both preparations have been used extensively for studies on energy transduction such as light-dependent cyclic electron transfer [2,3], proton translocation [4,5], photophosphorylation [6,7] and solute transport [1,5,8]. For the interpretation of the results obtained from these studies two questions about these membrane preparations have to be answered: 1. How is the membrane oriented in these vesicular structures (with respect to the cytoplasmic membrane in intact cells)? 2. Do both membrane preparations have comparable structural and functional properties? Qualitative information about the orientation of membrane vesicles and chromatophores has been obtained from studies on the localization of cytochrome c2 and ATPase activity, the direction of the light-induced protonmotive force and the protonmotive force-driven accumulation of amino acids and Ca2+ [ 1,5,8]. These studies revealed that membrane

Resistin Is More Abundant in Liver Than Adipose Tissue and Is Not Up-Regulated by Lipopolysaccharide

CONTEXT Resistin is an adipokine correlated with inflammatory markers and is predictive for cardiovascular diseases. There is evidence that serum resistin levels are elevated in obese patients; however, the role of resistin in insulin resistance and type 2 diabetes remains controversial. OBJECTIVE We addressed the question of whether inflammation may induce expression of resistin in organs involved in regulation of total body energy metabolism, such as liver and adipose tissue (AT). METHODS Human liver tissue, sc AT, and omentum were cultured in the absence/presence of lipopolysaccharide (LPS). The resistin and cytokine mRNA and protein expression levels were determined by real-time PCR, ELISA, and Multiplex Technology, respectively. The localization of resistin in human liver was analyzed by immunohistochemistry. RESULTS Resistin gene and protein expression was significantly higher in liver than in AT. Exposure of human AT and liver tissue in culture to LPS did not alter resistin concentration; however, concentrations of IL-1beta, IL-6, and TNFalpha were significantly increased in these tissues. In liver, resistin colocalizes with markers for Kupffer cells, for a subset of endothelial and fibroblast-like cells. CONCLUSIONS High level of resistin gene and protein expression in liver compared to AT implies that resistin should not be considered only as an adipokine in humans. LPS-induced inflammation does not affect resistin protein synthesis in human liver and AT. This suggests that elevated serum resistin levels are not indicative for inflammation of AT or liver in a manner similar to known inflammatory markers such as IL-1beta, IL-6, or TNFalpha.

The stability and functional properties of proteoliposomes mixed with dextran derivatives bearing hydrophobic anchor groups

Liposomes composed of Escherichia coli phospholipid were coated with polysaccharides bearing hydrophobic palmitoyl anchors. The effect on the stability of liposomes without or with integral membrane proteins was investigated. A high concentration of hydrophobized dextrans protected the liposomes against detergent degradation, decreased the fluidity of the membranes, prevented fusion of the liposomes and enhanced their stability. Proteoliposomes containing beef heart cytochrome-c oxidase and the lactose transport carrier of E. coli were similarly affected by coating with the dextrans. Under these conditions both membrane proteins were still active. Long-term stability of the coated liposomes was obtained only in the absence of the integral membrane proteins.

The Lactose Carrier of Escherichia Coli Functionally Incorporated in Rhodopseudomonas Sphaeroides Obeys the Regulatory Conditions of the Phototrophic Bacterium

Rhodopseudomonas sphaeroides was provided with the ability to transport lactose via conjugation with a strain of Escherichia coli bearing a plasmid containing the lactose operon (including the lac Y gene, coding for the lactose carrier or M protein) and subsequent expression of the lac operon in Rps. sphaeroides (Nano, F.E. and Kaplan, S. submitted). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton‐motive force. The results demonstrate that lactose transport is regulated by the rate of cyclic electron transfer in the same way as the endogenous transport systems.

The relation between electron transfer, proton-motive force and energy-consuming processes in cells of Rhodopseudomonas sphaeroides

Abstract In the phototrophic bacterium Rhodopseudomonas sphaeroides the two components of the proton-motive force (the ΔΨ and the ΔpH) were measured with ion-selective electrodes. Simultaneously, the rate of energy-consuming processes such as alanine transport or ATP-synthase activity was measured at increasing electron-transfer activity. The results indicate that the activity of the electron-transfer system determines the activity of the energy-consuming processes. The proton-motive force can increase or decrease with the activity of the electron-transfer chain depending on the experimental conditions. Consequently, under certain conditions the activity of secondary transport increases while the proton-motive force decreases. A fixed minimal rate of electron transfer is required before secondary solute transport can occur, independent of the magnitude of the proton-motive force. A threshold value of the proton-motive force for secondary solute transport is not required.

THERMOSTABILITY OF RESPIRATORY TERMINAL OXIDASES IN THE LIPID ENVIRONMENT

The effect of the lipid environment on the thermostability of three respiratory terminal oxidases was determined. Cytochrome-c oxidase from beef heart and Bacillus stearothermophilus were used as representative proteins from mesophilic and thermophilic origin, respectively. Quinol oxidase from the archaeon Sulfolobus acidocaldarius represented the model for a extreme thermoacidophilic enzyme. All three integral membrane proteins were tested for their thermal inactivation in detergent and after reconstitution in liposomes composed of phospholipids of Escherichia coli or tetraether lipids from S. acidocaldarius. When preincubated at 0 degrees C, all three enzymes exhibited biphasic thermal inactivation curves. Data could be analysed according to a two-state model that defines two conformations of the enzyme, differing in their thermostability. Monophasic inactivation curves were observed when the enzymes were preincubated at higher temperatures prior to thermal inactivation. Lipids rendered the beef-heart cytochrome-c oxidase and S. acidocaldarius quinol oxidase more thermostable as compared to detergent solution. In contrast, the B. stearothermophilus oxidase, an intrinsically thermostable enzyme, was as thermostable in detergent as in the reconstituted state. These data suggest that the lipid environment can be an important factor in the thermostability of membrane proteins.