Dicoumarol is frequently used as inhibitor of the detoxifying enzyme NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1). In order to test whether dicoumarol may also affect the cellular glutathione (GSH) metabolism, we have exposed cultured primary astrocytes to dicoumarol and investigated potential effects of this compound on the cell viability as well as on the cellular and extracellular contents of GSH and its metabolites. Incubation of astrocytes with dicoumarol in concentrations of up to 100 µM did not acutely compromise cell viability nor was any GSH consumption or GSH oxidation to glutathione disulfide (GSSG) observed. However, unexpectedly dicoumarol inhibited the cellular multidrug resistance protein (Mrp) 1-dependent export of GSH in a time- and concentration-dependent manner with half-maximal effects observed at low micromolar concentrations of dicoumarol. Inhibition of GSH export by dicoumarol was not additive to that observed for the known Mrp1 inhibitor MK571. In addition, dicoumarol inhibited also the Mrp1-mediated export of GSSG during menadione-induced oxidative stress and the export of the GSH–bimane-conjugate (GS–B) that had been generated in the cells after exposure to monochlorobimane. Half-maximal inhibition of the export of Mrp1 substrates was observed at dicoumarol concentrations of around 4 µM (GSH and GSSG) and 30 µM (GS–B). These data demonstrate that dicoumarol strongly affects the GSH metabolism of viable cultured astrocytes by inhibiting Mrp1-mediated export processes and identifies for the first time Mrp1 as additional cellular target of dicoumarol.
The review focuses on the multiple separating regimes that offers the free flow electrophoresis technique: free flow zone electrophoresis, isoelectric focusing, isotachophoresis, free flow step electrophoresis. Also, the feasibility to apply either interval or continuous flow electrophoresis is evaluated. The free flow zone electrophoresis regime is generally selected for the separation of cells, organelles and membranes while the other regimes find their largest fields of applications in the purification of proteins and peptides. The latter regimes present the highest resolution efficiency. Therefore, a large part of this review is devoted to the applicabilities of these different regimes to the purification of organelles and membrane vesicles at the preparative scale. Recent developments, both in instrumentation and procedures, are described. The major achievements in plant membrane fractionation obtained with free flow electrophoresis are outlined. The related procedures are both analytical and preparative: they separate tonoplast and plasma membrane simultaneously from the same homogenate, they discriminate for one type of membrane vesicles of opposite orientation, and process large quantities of membrane material by reason of the continuous flow mode. Recent advances using electromigration techniques that permit confirmation of the dynamic state of membranes, characterisation of complex membrane-dependent functions and discovery of new membrane-localised activities are presented. 相似文献
Cell separation by counterflow centrifugal elutriation (CCE) or free flow electrophoresis (FFE) is performed at lower frequency than cell cloning and antibody-dependent, magnetic or fluorescence-activated cell sorting. Nevertheless, numerous recent publications confirmed that these physical cell separation methods that do not include cell labeling or cell transformation steps, may be most useful for some applications. CCE and FFE have proved to be valuable tools, if homogeneous populations of normal healthy untransformed cells are required for answering scientific questions or for clinical transplantation and cells cannot be labeled by antibodies, because suitable antibodies are not available or because antibody binding to a cell surface would induce the cell reaction which should be investigated on purified cells or because antibodies bound to the surface hamper the use of the isolated cells. In addition, the methods are helpful for studying the biological reasons for, or effects of, changes in cell size and cellular negative surface charge density. Although the value of the methods was confirmed in recent years by a considerable number of important scientific results, activities to further develop and improve the instruments have, unfortunately, declined. 相似文献
Sampling of rat hepatic tissue for histomorphological analysis is usually performed in two different ways: either by killing the animals or by minilaparotomy.In this study we describe a percutaneous core biopsy technique which has been used on day 7, 14 and 21 after allogeneic rat liver transplantation (DA → LEW) in order to examine grafts for rejection in different treatment groups. Fifty-two liver biopsies were performed in 24 animals using a 16-gauge intravenous cannula. Forty-five provided usable specimens which were sufficient for both light or electron microscopy and immunohistochemical analyses to determine the degree of graft rejection. In 7 cases (13.5%) sampling was unsuccessful, especially on day 21 after transplantation, as the plastic cannula could not penetrate the hardened tissue. In 3 animals (5.8%) puncture was immediately followed by death due to perforation of the diaphragm or ether intoxication.In conclusion, this technique is a reliable method for providing ample tissue samples from the rat liver with a low risk of complications. 相似文献
The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans. 相似文献
The capacity of anoxygenic phototrophic bacteria to utilize aromatic hydrocarbons was investigated in enrichment cultures
with toluene. When mineral medium with toluene (provided in an inert carrier phase) was inoculated with activated sludge and
incubated under infrared illumination (> 750 nm), a red-to-brownish culture developed. Agar dilution series indicated the
dominance of two types of phototrophic bacteria. One type formed red colonies, had rod-shaped cells with budding division,
and grew on benzoate but not on toluene. The other type formed yellow-to-brown colonies, had oval cells, and utilized toluene
and benzoate. One strain of the latter type, ToP1, was studied in detail. Sequence analysis of the 16S rRNA gene and DNA-DNA
hybridization indicated an affiliation of strain ToP1 with the species Blastochloris sulfoviridis, a member of the α-subclass of Proteobacteria. However, the type strain (DSM 729) of Blc. sulfoviridis grew neither on toluene nor on benzoate. Light-dependent consumption of toluene in the presence of carbon dioxide and formation
of cell mass by strain ToP1 were demonstrated in quantitative growth experiments. Strain ToP1 is the first phototrophic bacterium
shown to utilize an aromatic hydrocarbon. In the supernatant of toluene-grown cultures and in cell-free extracts incubated
with toluene and fumarate, the formation of benzylsuccinate was detected. These findings indicate that the phototrophic bacterium
activates toluene anaerobically by the same mechanism that has been reported for denitrifying and sulfate-reducing bacteria.
The natural abundance of phototrophic bacteria with the capacity for toluene utilization was examined in freshwater habitats.
Counting series revealed that up to around 1% (1.8 × 105 cells per gram dry mass of sample) of the photoheterotrophic population cultivable with acetate grew on toluene.
Received: 29 March 1999 / Accepted: 1 July 1999 相似文献