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Siegl T.,Albert Ludwigs University of Freiburg | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research Saarland
Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology | Year: 2012

This review provides an overview of new technologies for DNA manipulations in actinomycetes exploiting recombinogenic engineering (Flp-FRT, Cre-loxP, Dre-rox, Tn5, GusA and I-SceI systems). We will describe some new vectors recently developed for engineering of complex phenotypes in actinomycetes. Several site-specific recombinases, transposons, reporter genes and I-SceI endonuclease have been utilized for genome manipulation in actinomycetes. Novel molecular tools will help to overcome many technical difficulties and will encourage new efforts to address the function of actinomy-cete genes. © Springer Science+Business Media B.V. 2012.

Siegl T.,Albert Ludwigs University of Freiburg | Tokovenko B.,Helmholtz Institute for Pharmaceutical Research Saarland | Myronovskyi M.,Helmholtz Institute for Pharmaceutical Research Saarland | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research Saarland
Metabolic Engineering | Year: 2013

We developed a synthetic promoter library for actinomycetes based on the -10 and -35 consensus sequences of the constitutive and widely used ermEp1 promoter. The sequences located upstream, in between and downstream of these consensus sequences were randomised using degenerate primers and cloned into an integrative plasmid upstream of the gusA reporter gene. Using this system, we created promoters with strengths ranging from 2% to 319% compared with ermEp1. The strongest synthetic promoter was used in a proof-of-principle approach to achieve the overexpression of a natural type III polyketide synthase. We observed high correlation between the number of gusA reporter gene RNA-Seq reads and the GusA reporter protein activity, indicating that GusA is indeed a transcription-level reporter system. © 2013 Elsevier Inc.

Lehmann A.D.,University of Bern | Daum N.,Helmholtz Institute for Pharmaceutical Research Saarland | Bur M.,Helmholtz Institute for Pharmaceutical Research Saarland | Lehr C.-M.,Helmholtz Institute for Pharmaceutical Research Saarland | And 2 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2011

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm2) was higher than in A549 (172 ± 59 Ω cm2) but similar to 16HBE14o- cells (1469 ± 156 Ω cm2). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm2) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm2) and 16HBE14o- (558 ± 267 Ω cm2). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall. © 2010 Elsevier B.V. All rights reserved.

Negri M.,Saarland University | Negri M.,Helmholtz Institute for Pharmaceutical Research Saarland | Recanatini M.,University of Bologna | Hartmann R.W.,Saarland University | Hartmann R.W.,Helmholtz Institute for Pharmaceutical Research Saarland
PLoS ONE | Year: 2010

Background: Bisubstrate enzymes, such as 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), exist in solution as an ensemble of conformations. 17β-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment. Methodology/Principal Findings: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17β-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step. Conclusions/Significance: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the βFαG′-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17β-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17β-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17β-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis. © 2010 Negri et al.

Haupenthal J.,Helmholtz Institute for Pharmaceutical Research Saarland | Husecken K.,Helmholtz Institute for Pharmaceutical Research Saarland | Negri M.,Helmholtz Institute for Pharmaceutical Research Saarland | Maurer C.K.,Helmholtz Institute for Pharmaceutical Research Saarland | And 2 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2012

In recent decades, quantitative transcription assays using bacterial RNA polymerase (RNAP) have been performed under widely diverse experimental conditions. We demonstrate that the template choice can influence the inhibitory potency of RNAP inhibitors. Furthermore, we illustrate that the sigma factor (σ70) surprisingly increases the transcription efficiency of templates with nonphysiological nonprokaryotic promoters. Our results might be a useful guideline in the early stages of using RNAP for drug discovery. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Neumeyer A.,Saarland University | Bukowski M.,Leibniz Institute for New Materials | Veith M.,Leibniz Institute for New Materials | Lehr C.-M.,Saarland University | And 3 more authors.
Nanomedicine: Nanotechnology, Biology, and Medicine | Year: 2011

Because nanoparticles are promising tools in drug delivery, quantification of their cellular binding and uptake is an emerging question. Therefore, rhodamine B isothiocyanate-labeled silica nanoparticles with different sizes and surface modifications were investigated concerning their uptake in Caco-2 cells. Flow cytometry studies exhibited a size- and time-dependent association for unmodified nanoparticles (50 and 77 nm), whereas larger particles (94 nm) and polyethylene glycol-modified nanoparticles showed no cellular interaction. A second approach dealt with particles with adsorbed propidium iodide (PI) to distinguish between internalized and adsorbed nanoparticles. These particles only give a fluorescence signal when associated with nucleic acids inside the cell, whereas particles adsorbed to the outer cell surface are not detected. PI-labeled nanoparticles (21 nm) showed a time-dependent uptake, exhibiting a signal in the cytoplasm but less in the nucleus. These novel PI-labeled nanoparticles in combination with flow cytometry are innovative tools for the quantification of nanoparticulate uptake. © 2011 Elsevier Inc.

Van Koppen C.J.,ElexoPharm GmbH | Hartmann R.W.,ElexoPharm GmbH | Hartmann R.W.,Saarland University | Hartmann R.W.,Helmholtz Institute for Pharmaceutical Research Saarland
Expert Opinion on Therapeutic Patents | Year: 2015

Introduction: About 2% of the Western world population suffer from chronic wounds, resulting from underlying disorders (e.g., diabetes, excessive pressure, vascular insufficiencies and vasculitis), with a significant adverse effect on Quality of Life. Despite high incidence and economic burden, management of chronic wounds is still far from effective and novel therapies are in urgent need. Wound healing is a dynamic process of transient expression, function and clearance of mediators, enzymes and cell types. Failure to initiate, terminate or regulate leads to pathologic wound healing.Areas covered: The present review discusses patents of the seven most promising classes of biological agents, mostly published in 2009-2014 (CYP11B1 inhibitors, peptide growth factors, prolyl-4-hydroxylase and matrix metalloproteinase inhibitors, bone marrow-derived mesenchymal stem cells, elastase and connexin43 inhibitors). Relevant information from peer-reviewed journals is also presented.Expert Opinion: The aforementioned biological agents have different mechanisms of action, and considering the multifactorial pathogenesis of chronic wounds, they hold promise in treating chronic wounds. However, as administration of a certain biological agent may be beneficial in an early phase, it may slow down wound healing in a later phase. Basic and clinical research on chronic wound healing should therefore investigate the efficacy of these agents, alone and in concert, during the consecutive phases of wound healing. © Informa UK, Ltd.

Myronovskyi M.,Helmholtz Institute for Pharmaceutical Research Saarland | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research Saarland
Applied Microbiology and Biotechnology | Year: 2013

The rational modification of the actinomycetes genomes has a variety of applications in research, medicine, and biotechnology. The use of site-specific recombinases allows generation of multiple mutations, large DNA deletions, integrations, and inversions and may lead to significant progress in all of these fields. Despite their huge potential, site-specific recombinase-based technologies have primarily been used for simple marker removal from a chromosome. In this review, we summarise the site-specific recombination approaches for genome engineering in various actinomycetes. © 2013 Springer-Verlag Berlin Heidelberg.

Salomon J.J.,Trinity College Dublin | Muchitsch V.E.,Trinity College Dublin | Gausterer J.C.,Trinity College Dublin | Schwagerus E.,Trinity College Dublin | And 4 more authors.
Molecular Pharmaceutics | Year: 2014

The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP + uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm2, after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors. In transport studies, Rh123 exhibited net secretion, which again was inhibitable by bona fide P-gp modulators. The uptake of ASP+ was time-and temperature-dependent with Km = 881.2 ± 195.3 μM and Vmax = 2.07 ± 0.26 nmol/min/mg protein. TEA, amantadine, quinidine, and verapamil significantly inhibited ASP+ uptake into NCl-H441 cells, whereas the effect of d-and l-carnitine and ergothioneine, two OCTN substrates, was less pronounced. NCl-H441 cells are the first cell line of human distal lung epithelial origin with the ability to form monolayers with appreciable barrier properties. Moreover, drug transporter expression and activity in NCl-H441 cells was consistent with what has been reported for human alveolar epithelial cells in primary culture. © 2014 American Chemical Society.

Bilyk B.,Helmholtz Institute for Pharmaceutical Research Saarland | Luzhetskyy A.,Helmholtz Institute for Pharmaceutical Research Saarland
Applied Microbiology and Biotechnology | Year: 2014

The φC31-encoded recombination system has become a widely used tool for genetic analysis of streptomycetes, gene therapy and generation of transgenic animals. However, the application of this system, even in the context of its natural host genus, Streptomyces, may require a specific approach for each species. In this study, we have identified a novel pseudo-attB site, called pseB4, for integration of vectors using the φC31 system. More than 90 % of clones contained two copies of pSET152- or pOJ436-based cosmids, after their introduction into S. albus. The efficiency of the integration of φC31-based vectors into pseB4 is therefore comparable to that of the integration into attB. Moreover, in contrast with integration into the native attB, integration into pseB4 is not polar and does not require a complementary sequence in the TT-core region. Furthermore, an analysis of conjugation frequency revealed mutual inhibition of plasmid integration into either site when both the attB and pseB4 sites were present in the genome. © 2014 Springer-Verlag.

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