Publikationen

Reaction of ligand LH2 (4,5-bis[carboxymethylthio]-1,3-dithiol-2-thione) with neodymium silyl-amide (Nd[N(TMS)2]3; TMS= -SiMe3), in a ratio 2:1, yields a neodymium-dithiolene-carboxylato complex (1) (Nd(LH)L). Similarly, reaction of 2 equivalents of L'H2 (4,5-bis[2-hydroxyethyl)thio]-1,3-dithiol-2-thione) and one equivalent of neodymium silyl-amide (Nd[N(TMS)2]3) allowed the isolation of complex 2, with a ligand:metal ratio of 3:2. ATR-IR spectrum of 1 displays a broad band characteristic of an OH group showing that one carboxylate group remains protonated. Emission spectrum of complex 1 under excitation in the visible region (at 360 nm i.e. on the ligand) displayed typical emission bands of the Nd3+, showing that energy transfer from the ligand to the lanthanide was achieved (i.e. "antenna effect"). No significant quenching from the remaining -OH group was detected. In the case of complex 2, the main emission bands characteristic of the Nd3+ ion have been observed, by excitation at 495 nm.

The reaction between [(Ph2Si)2O3]4[Al(OH)]4 (1) or [(Ph2Si)2O3]4[Al(OLi)]4 (2) with sodium ethoxide, or lithium hydroxide in presence of CuI·H2O leads to the formation of new alumopolysiloxane compounds. Indeed, transformations of 1 under the partial incorporation of the reactants are found giving rise to new heteroleptic inorganic macrocycles. The molecular structure of [(Ph2Si)2O3]4[Al(ONa)]2[Al(OH)(NaOEt)]2·2Et2O (3) and [(Ph2Si)2O3]4[Al(OLi)]2[Al(OH)(LiOH)]2·2Et2O·2THF (4) have been determined by single-X-ray diffraction analysis. Both alumosiloxanes 3 and 4 are constituted by a twelve-membered ring.

Heterogeneous thin films may be beneficial for sensoring devices. The electrical conductivity of nanoscale metallic particles being embedded in a matrix of non conducting material should exhibit higher sensitivity to mechanical stress and strain compared to homogeneous films. The production of heterogeneous films may follow different routes. This paper describes the attempt to embed Ag nanoclusters emitted from a gas aggregation cluster source into a growing matrix of alumina originating from sputter sources. The characteristics of the cluster source are first resumed, with their mean masses ranging from approx. 1000 to 100,000 atoms per cluster. The expelled and soft landed clusters are extensively examined by transmission electron microscopy verifying their crystalline form. Yet the use of a radio frequency driven sputter source for the embed material destroys and annihilates the Ag clusters even at very low sputter power. If a reactive direct current sputter process is performed within an oxidising sputter gas instead, the Ag clusters are oxidised to different oxides, but they survive as crystalline entities as verified by X-ray diffraction investigations. A simple subsequent heat treatment reduces the Ag oxides to metallic Ag clusters.

Background: Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain. Results: Enzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-Dglucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 μM-10 μM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges. Conclusion: Provided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types.

It is widely accepted that the functional and morphological differentiation of cells is initiated and determined by the interaction of molecules of the extracellular matrix and adhesion molecules of the cell membrane. To assess the influence of the underlying matrix on the characteristics of cells, enterocyte-like Caco-2 cells were cultivated on substrates commonly used for cell culture as well as on glass coated with hydrophobic layers. Providing the same starting conditions for growth, the parameters investigated on pre-confluent Caco-2 cells were the number of adhering cells, the proliferative activity and the degree of differentiation indicated by the expression of three brush border enzymes. Whereas tissue culture treated polystyrene elicited highest rates of adhesion, proliferation, and differentiation, even glass altered the pattern of brush border enzyme expression. The hydrophobic surfaces strongly decreased the adhesion and the proliferation but the surviving cells exhibited a pronounced higher degree of differentiation. Interestingly, each subtype of hydrophobic matrix triggered a different pattern of brush border enzyme expression. Thus, the development of a certain phenotype of a cell can not only be triggered by certain components of the extracellular matrix but also by artificially prepared surface coatings of the underlying matrix. In the future it seems to be feasible that cells can be programmed by tailoring the surface of the underlying substrate.

For optimal compatibility with biopharmaceutical manufacturing and gene therapy, heterologous transgene control systems must be responsive to side-effect-free physiologic inducer molecules. The arginine-inducible interaction of the ArgR repressor and the ArgR-specific ARG box, which synchronize arginine import and synthesis in the intracellular human pathogen Chlamydia pneumoniae, was engineered for arginine-regulated transgene (ART) expression in mammalian cells. A synthetic arginine-responsive transactivator (ARG), consisting of ArgR fused to the Herpes simplex VP16 transactivation domain, reversibly adjusted transgene transcription of chimeric ARG box-containing mammalian minimal promoters (PART) in an arginine-inducible manner. Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology. ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types. Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses. Using a physiologic inducer, such as the amino acid L-arginine, to control heterologous transgenes in a seamless manner which is devoid of noticeable metabolic interference will foster novel opportunities for precise expression dosing in future gene therapy scenarios as well as the manufacturing of difficult-to-produce protein pharmaceuticals. © 2007 The Author(s).

Excessive use of antibiotics in veterinary medicine and as growth promoters in stock farming has been associated with the dramatically increasing prevalence of multidrug-resistant human pathogenic bacteria. European community legislators have therefore restricted the veterinary use of antibiotics and banned them as growth-promoting food additives in stock breeding (1831/2003/EC). The monitoring of such legislation requires technology for precise and straightforward on-site quantification of antibiotics in farm samples and food products without the need for extensive laboratory equipment and trained personnel. Capitalizing on bacterial transcriptional regulators (TetR, PIP, E), which are dose-dependently released from their cognate operators (tetO, PIR, ETR) upon binding of specific classes of antibiotics (tetracycline, streptogramins, macrolides) we have designed an easy-to-handle dipstick-based assay for detection of antibiotic levels in serum, meat and milk whose detection limits are up to 40-fold below licensed threshold values. The generic dipstick consists of either nitrocellulose, nylon or polyvinylidenfluorid (PVDF) membrane strips coated with streptavidin and immobilized biotinylated operator DNA, which acts as capture DNA to bind hexa-histidine (His6)-tagged bacterial biosensors. Antibiotics present in specific samples triggered the dose-dependent release of the capture DNA-biosensor interaction, which, after dipping into two different solutions, results in a correlated conversion of a chromogenic substrate by a standard His6-targeted enzyme complex. This can be quantified by comparison of the dipstick to a standardized color scale or by assessing the terminal solution at 450 nm. As demonstrated using serum, meat and milk samples spiked with 14 different antibiotics, the dipstick technology provided sensitive detection in a rapid assay format, and could be employed to monitor non-authorized use of antibiotics and to discover novel antibiotics. © 2006 Elsevier B.V. All rights reserved.

Although adjustable transgene expression systems are considered essential for future therapeutic and biopharmaceutical manufacturing applications, the currently available transcription control modalities all require side-effect-prone inducers such as immunosupressants, hormones and antibiotics for fine-tuning. We have designed a novel mammalian transcription-control system, which is reversibly fine-tuned by non-toxic vitamin H (also referred to as biotin). Ligation of vitamin H, by engineered Escherichia coli biotin ligase (BirA), to a synthetic biotinylation signal fused to the tetracycline-dependent transactivator (tTA), enables heterodimerization of tTA to a streptavidin-linked transrepressor domain (KRAB), thereby abolishing tTA-mediated transactivation of specific target promoters. As heterodimerization of tTA to KRAB is ultimately conditional upon the presence of vitamin H, the system is vitamin H responsive. Transgenic Chinese hamster ovary cells, engineered for vitamin H-responsive gene expression, showed high-level, adjustable and reversible production of a human model glycoprotein in bench-scale culture systems, bioreactor-based biopharmaceutical manufacturing scenarios, and after implantation into mice. The vitamin H-responsive expression systems showed unique band pass filter-like regulation features characterized by high-level expression at low (0-2 nM biotin), maximum repression at intermediate (100-1000 nM biotin), and high-level expression at increased (> 100 000 nM biotin) biotin concentrations. Sequential ON-to-OFF-to-ON, ON-to-OFF and OFF-to-ON expression profiles with graded expression transitions can all be achieved by simply increasing the level of a single inducer molecule without exchanging the culture medium. These novel expression characteristics mediated by an FDA-licensed inducer may foster advances in therapeutic cell engineering and manufacturing of difficult-to-produce protein therapeutics. © 2007 The Author(s).

Inducible transgene control systems have been instrumental to gene therapy, biopharmaceutical manufacturing, drug discovery, synthetic biology and functional genomic research. The most widely used heterologous gene regulation systems are responsive to antibiotics of the tetracycline, streptogramin and macrolide classes. Although these antibiotics are clinically licensed, concerns about the emergence of resistant bacteria, side-effects in animal studies, and economic considerations associated with clearance of antibiotics in biopharmaceutical manufacturing, have limited the use of heterologous transgene control modalities to basic research activities. We have therefore designed a strategy to convert antibiotic-responsive transcription factors into gene regulation systems responsive to non-toxic biotin, also known as vitamin H. Constitutive ligation of biotin to the Avitag-containing VP16 transactivation domain by the Escherichia coli biotin ligase BirA enables heterodimerization with tetracycline- (TetR), streptogramin- (Pip), and macrolide- (E) dependent repressors fused to streptavidin, which creates synthetic transactivators able to activate specific promoters (PhCMV*-1, PPIR, PETR). We have demonstrated (i) that exogenous biotin (40 nM) can induce heterologous transgene expression in a biotin- (serum-) free culture environment (biotin-dependent heterodimerization of transactivator); (ii) that excess biotin (above 200 μM) gradually represses transgene expression in a biotin- (serum-) containing environment (saturation of streptavidin by excess biotin prevents heterodimerization of the transactivator); and (iii) that avidin can sequestrate endogenous biotin in serum-containing cultures and so repress transgene expression in a dose-dependent manner. In addition, by engineering all off the components required for biotin-controlled transgene expression (Avitag-VP16, repressor-streptavidin, BirA) into a tricistronic (lenti)vector configuration, it was possible to transfect (transduce) a variety of mammalian cell lines and primary cells and enable biotin-controlled transgene expression in a simple and straightforward manner. The conversion of generic antibiotic-responsive transcription control modalities into systems adjustable by non-toxic vitamin H may foster novel advances in reprogramming of mammalian cells and production of difficult-to-produce protein pharmaceuticals. © 2007 Elsevier B.V. All rights reserved.