The choreography of multidrug export

Multidrug transporters have a crucial role in causing the drug resistance that can arise in infectious micro-organisms and tumours. These integral membrane proteins mediate the export of a broad range of unrelated compounds from cells, including antibiotics and anticancer agents, thus reducing the concentration of these compounds to subtoxic levels in target cells. In spite of intensive research, it is not clear exactly how multidrug transporters work. The present review focuses on recent advancements in the biochemistry and structural biology of bacterial and human multidrug ABC (ATP-binding cassette) transporters. These advancements point to a common mechanism in which polyspecific drug-binding surfaces in the membrane domains are alternately exposed to the inside and outside surface of the membrane in response to the ATP-driven dimerization of nucleotide-binding domains and their dissociation following ATP hydrolysis.     

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5β1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.     

A high-throughput method for membrane protein solubility screening: the ultracentrifugation dispersity sedimentation assay

One key to successful crystallization of membrane proteins is the identification of detergents that maintain the protein in a soluble, monodispersed state. Because of their hydrophobic nature, membrane proteins are particularly prone to forming insoluble aggregates over time. This nonspecific aggregation of the molecules reduces the likelihood of the regular association of the protein molecules essential for crystal lattice formation. Critical buffer components affecting the aggregation of membrane proteins include detergent choice, salt concentration, and presence of glycerol. The optimization of these parameters is often a time- and protein-consuming process. Here we describe a novel ultracentrifugation dispersity sedimentation (UDS) assay in which ultracentrifugation of very small (5 microL) volumes of purified, soluble membrane protein is combined with SDS-PAGE analysis to rapidly assess the degree of protein aggregation. The results from the UDS method correlate very well with established methods like size-exclusion chromatography (SEC), while consuming considerably less protein. In addition, the UDS method allows rapid screening of detergents for membrane protein crystallization in a fraction of the time required by SEC. Here we use the UDS method in the identification of suitable detergents and buffer compositions for the crystallization of three recombinant prokaryotic membrane proteins. The implications of our results for membrane protein crystallization prescreening are discussed.     

The theory of organism and the culturalist foundation of biology

Summary After the disappearance of organism was diagnosed, the discussion about the role of a theory of organism in biology is characterised by a significant contradiction. On the one hand, the importance of a theory of organism is stated. Particularly developmental biology demands organism-centred approaches as a basis for conceptual integration. On the other hand, several modern biological disciplines such as genetics and molecular biology simply don’t need a theory of organism for their work. Consequently, the determination of the status of the organism and its relevance for biology at all is an unsolved problem. In order to clarify the methodological status of the organism in biology we start with the reconstruction of three important propositions. A life oriented approach and a hierarchy concept - which both are from a neo-Darwinian origin - are confronted with a structuralist approach of organism, that can be characterised as a non-Darwinist approach. Our own attempt for the solution of the organism problem applies the tools of culturalist methodology. In accordance to this pragmatic approach, the term organism is introduced as a concept of notion. A constructional morphological case study exemplifies the applicability of this concept. From the culturalist point of view a methodological foundation of biology can be achieved, that provides a consistent basis for a comprehensive integration of biological knowledge.     

Polyvinylamine‐coated polyester fibers as a carrier matrix for the immobilization of peroxidases

Biocatalytic transformations that employ immobilized enzymes become increasingly important for industrial applications. Synthetic or natural textile fiber materials such as polyester, polyamide or viscose are support materials that are comparatively inexpensive. Contrary to traditional support materials, their flexibility enables their use in reactors of any geometry and a fast and residue‐free removal from batch reactors. In this study a permanently immobilized peroxidase (Baylase®) has been investigated on polyester felt as a solid support as a new heterogeneous catalyst system. The polyester felt was functionalized by coating with polyvinylamine and subsequent activation with glutaraldehyde as a crosslinking agent. The enzyme load on the textile surface, the activity of the immobilized protein after repeated use as well as the storage stability was evaluated. Scanning electron micrographs and UV Vis spectroscopy made it possible to verify the enzyme immobilization on the textile surface. Furthermore, the load of immobilized peroxidase was determined by ICP OE spectrometry to be 9–12 mg per gram of textile. The activity of immobilized Baylase® remained high over 35 reaction cycles and a storage period of 8 weeks.     

The β-lactam-sensitive d,d-carboxypeptidase activity of Pbp4 controls the l,d and d,d transpeptidation pathways in Corynebacterium jeikeium

Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to β-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of β-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d , d and l , d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt_Cjk1, a member of the active site cysteine l , d -transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of β-lactam, the PBPs and the l , d -transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt_Cjk1 and Pbp2C were not inhibited by ampicillin, the participation of the l , d -transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt_Cjk1 for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt_Cjk1 was produced by an ampicillin-sensitive d , d -carboxypeptidase (Pbp4_Cjk). Acquisition and mutational alterations of pbp2C accounted for high-level β-lactam resistance in C. jeikeium .     

Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faecium

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.     

Trophic consequences for riverine cyprinid fishes of angler subsidies based on marine‐derived nutrients

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Binding of inositol phosphates to arrestin.

Arrestin binds to phosphorylated rhodopsin in its light-activated form (metarhodopsin II), blocking thereby its interaction with the G-protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin-rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5-tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 microM and 5 microM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.