Ethylbenzene dehydrogenase, a novel hydrocarbon-oxidizing molybdenum/iron-sulfur/heme enzyme

The initial enzyme of ethylbenzene metabolism in denitrifying Azoarcus strain EbN1, ethylbenzene dehydrogenase, was purified and characterized. The soluble periplasmic enzyme is the first known enzyme oxidizing a nonactivated hydrocarbon without molecular oxygen as cosubstrate. It is a novel molybdenum/iron-sulfur/heme protein of 155 kDa, which consists of three subunits (96, 43, and 23 kDa) in an αβγ structure. The N-terminal amino acid sequence of the α subunit is similar to that of other molybdenum proteins such as selenate reductase from the related speciesThauera selenatis. Ethylbenzene dehydrogenase is unique in that it oxidizes the hydrocarbon ethylbenzene, a compound without functional groups, to (S)-1-phenylethanol. Formation of the product was evident by coupling to an enantiomer-specific (S)-1-phenylethanol dehydrogenase from the same organism. The apparent K m of the enzyme for ethylbenzene is very low at <2 μm. Oxygen does not affect ethylbenzene dehydrogenase activity in extracts but inactivates the purified enzyme, if the heme b cofactor is in the reduced state. A variant of ethylbenzene dehydrogenase exhibiting significant activity also with the homolog n-propylbenzene was detected in a relatedAzoarcus strain (PbN1)

XL4C4D - Adding the Graph Transformation Language XL to CINEMA 4D

A plug-in for the 3D modeling application CINEMA 4D is presented which allows to use the graph transformation language XL to transform the 3D scene graph of CINEMA 4D. XL extends Java by graph query and rewrite facilities via a data model interface, the default rewrite mechanism is that of relational growth grammars which are based on parallel single-pushout derivations. We illustrate the plug-in at several examples, some of which make use of advanced 3D features

Realization and Extension of the Xfrog Approach for Plant Modelling in the Graph-Grammar Based Language XL

Two well-known approaches for modelling virtual vegetation are grammar-based methods (L-systems) and the Xfrog method, which is based on graph transformations expanding "multiplier" nodes. We show that both approaches can be unified in the framework of "relational growth grammars", a variant of parallel graph grammars. We demonstrate this possibility and the synergistic benefits of the combination of both methods at simple plant models which were processed using our open-source software GroIMP

Entwurf und Implementation einer auf Graph-Grammatiken beruhenden Sprache zur Funktions-Struktur-Modellierung von Pflanzen

Increasing biological knowledge requires more and more elaborate methods to translate the knowledge into executable model descriptions, and increasing computational power allows to actually execute these descriptions. Such a simulation helps to validate, extend and question the knowledge. For plant modelling, the well-established formal description language of Lindenmayer systems reaches its limits as a method to concisely represent current knowledge and to conveniently assist in current research. On one hand, it is well-suited to represent structural and geometric aspects of plant models - of which units is a plant composed, how are these connected, what is their location in 3D space -, but on the other hand, its usage to describe functional aspects - what internal processes take place in the plant structure, how does this interact with the structure - is not as convenient as desirable. This can be traced back to the underlying representation of structure as a linear chain of units, while the intrinsic nature of the structure is a tree or even a graph. Therefore, we propose to use graphs and graph grammars as a basis for plant modelling which combines structural and functional aspects. In the first part of this thesis, we develop the necessary theoretical framework. Starting with a presentation of the state of the art concerning Lindenmayer systems and graph grammars, we develop the formalism of relational growth grammars as a variant of graph grammars. We show that this formalism has a natural embedding of Lindenmayer systems which keeps all relevant properties, but represents branched structures directly as axial trees and not as linear chains with indirect encoding of branches. In the second part, we develop the main practical result, the XL programming language as an extension of the Java programming language by very general rule-based features. Short examples illustrate the application of the new language features. We describe the built-in pattern matching algorithm of the implemented run-time system for the XL programming language, and we sketch a possible implementation of an XL compiler. The third part is an application of relational growth grammars and the XL programming language. We show how the general XL interfaces can be customized for relational growth grammars. On top of this customization, several examples from a variety of disciplines demonstrate the usefulness of the developed formalism and language to describe plant growth, especially functional-structural plant models, but also artificial life, architecture or interactive games. Some examples operate on custom graphs like XML DOM trees or scene graphs of commercial 3D modellers, while the majority uses the 3D modelling platform GroIMP, a software developed in conjunction with this thesis. The appendix gives an overview of the GroIMP software. The practical usage of its plug-in for relational growth grammars is also illustrated.Das zunehmende Wissen über biologische Prozesse verlangt nach geeigneten Methoden, es in ausführbare Modelle zu übersetzen, und die zunehmende Rechenleistung der Computer ermöglicht es, diese Modelle auch tatsächlich auszuführen. Solche Simulationen dienen zur Validierung, Erweiterung und Hinterfragung des Wissens. Speziell für die Pflanzenmodellierung wurden Lindenmayer-Systeme mit Erfolg eingesetzt, jedoch stoßen diese bei aktuellen Modellierungsproblemen und Forschungsvorhaben an ihre Grenzen. Zwar sind sie gut geeignet, Pflanzenstruktur und Geometrie abzubilden - aus welchen Einheiten setzt sich eine Pflanze zusammen, wie sind diese verbunden, wie ist ihre räumliche Lage -, aber die lineare Datenstruktur erschwert die Integration von Funktionsmodellen, welche Prozesse innerhalb der verzweigten Struktur und des beanspruchten Raumes beschreiben. Daher wird in dieser Arbeit vorgeschlagen, anstelle der linearen Stuktur Graphen und Graph-Grammatiken als Grundlage für die kombinierte Funktions-Struktur-Modellierung von Pflanzen zu verwenden. Im ersten Teil der Dissertation wird der theoretische Unterbau entwickelt. Nach einer Vorstellung des aktuellen Wissensstandes auf dem Gebiet der Lindenmayer-Systeme und Graph-Grammatiken werden relationale Wachstumsgrammatiken eingeführt, die auf bekannten Mechanismen für parallele Graph-Grammatiken aufbauen und Lindenmayer-Systeme als Spezialfall enthalten, dabei jedoch verzweigte Strukturen direkt als axiale Bäume darstellen. Zur praktischen Anwendung wird im zweiten Teil die Programmiersprache XL entwickelt, die Java um allgemein gehaltene Sprachkonstrukte für Graph-Grammatiken erweitert. Kurze Beispiele zeigen die Anwendung der neuen Sprachmerkmale. Der Algorithmus zur Mustersuche wird erläutert, und die Implementation des XL-Compilers wird vorgestellt. Im dritten Teil werden mögliche Anwendungen relationaler Wachstumsgrammatiken aufgezeigt. Dazu werden zunächst die allgemeinen XL-Schnittstellen für relationale Wachstumsgrammatiken konkretisiert, um dieses System dann für Modelle aus verschiedenen Bereichen zu nutzen, darunter Funktions-Struktur-Modelle von Pflanzen, Künstliches Leben, Architektur und interaktive Spiele. Einige Beispiele nutzen spezifische Graphen wie XML-DOM-Bäume oder Szenengraphen kommerzieller 3D-Modellierprogramme, aber der überwiegende Teil baut auf der 3D-Plattform GroIMP auf, die zusammen mit dieser Dissertation entwickelt wurde. Im Anhang wird die Software GroIMP kurz vorgestellt und ihre praktische Anwendung für relationale Wachstumsgrammatiken erläutert

Ludo: A Case Study for Graph Transformation Tools

In this paper we describe the Ludo case, one of the case studies of the AGTIVE 2007 Tool Contest (see [22]). After summarising the case description, we give an overview of the submitted solutions. In particular, we propose a number of dimensions along which choices had to be made when solving the case, essentially setting up a solution space; we then plot the spectrum of solutions actually encountered into this solution space. In addition, there is a brief description of the special features of each of the submissions, to do justice to those aspects that are not distinguished in the general solution space

Integrative analysis of the heat shock response in Aspergillus fumigatus

<p>Abstract</p> <p>Background</p> <p><it>Aspergillus fumigatus </it>is a thermotolerant human-pathogenic mold and the most common cause of invasive aspergillosis (IA) in immunocompromised patients. Its predominance is based on several factors most of which are still unknown. The thermotolerance of <it>A. fumigatus </it>is one of the traits which have been assigned to pathogenicity. It allows the fungus to grow at temperatures up to and above that of a fevered human host. To elucidate the mechanisms of heat resistance, we analyzed the change of the <it>A. fumigatus </it>proteome during a temperature shift from 30°C to 48°C by 2D-fluorescence difference gel electrophoresis (DIGE). To improve 2D gel image analysis results, protein spot quantitation was optimized by missing value imputation and normalization. Differentially regulated proteins were compared to previously published transcriptome data of <it>A. fumigatus</it>. The study was augmented by bioinformatical analysis of transcription factor binding sites (TFBSs) in the promoter region of genes whose corresponding proteins were differentially regulated upon heat shock.</p> <p>Results</p> <p>91 differentially regulated protein spots, representing 64 different proteins, were identified by mass spectrometry (MS). They showed a continuous up-, down- or an oscillating regulation. Many of the identified proteins were involved in protein folding (chaperones), oxidative stress response, signal transduction, transcription, translation, carbohydrate and nitrogen metabolism. A correlation between alteration of transcript levels and corresponding proteins was detected for half of the differentially regulated proteins. Interestingly, some previously undescribed putative targets for the heat shock regulator Hsf1 were identified. This provides evidence for Hsf1-dependent regulation of mannitol biosynthesis, translation, cytoskeletal dynamics and cell division in <it>A. fumigatus</it>. Furthermore, computational analysis of promoters revealed putative binding sites for an AP-2alpha-like transcription factor upstream of some heat shock induced genes. Until now, this factor has only been found in vertebrates.</p> <p>Conclusions</p> <p>Our newly established DIGE data analysis workflow yields improved data quality and is widely applicable for other DIGE datasets. Our findings suggest that the heat shock response in <it>A. fumigatus </it>differs from already well-studied yeasts and other filamentous fungi.</p

Identification of PARP-1, Histone H1 and SIRT-1 as new regulators of breast cancer-related aromatase promoter I.3/II

Paracrine interactions between malignant estrogen receptor positive (ER+) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD+/NADH-ratio in BAFs. This suggests that cellular NAD+/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD+/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers

Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter

<p>Abstract</p> <p>Background</p> <p><it>Aspergillus fumigatus </it>is a mold responsible for the majority of cases of aspergillosis in humans. To survive in the human body, <it>A. fumigatus </it>must adapt to microenvironments that are often characterized by low nutrient and oxygen availability. Recent research suggests that the ability of <it>A. fumigatus </it>and other pathogenic fungi to adapt to hypoxia contributes to their virulence. However, molecular mechanisms of <it>A. fumigatus </it>hypoxia adaptation are poorly understood. Thus, to better understand how <it>A. fumigatus </it>adapts to hypoxic microenvironments found <it>in vivo </it>during human fungal pathogenesis, the dynamic changes of the fungal transcriptome and proteome in hypoxia were investigated over a period of 24 hours utilizing an oxygen-controlled fermenter system.</p> <p>Results</p> <p>Significant increases in transcripts associated with iron and sterol metabolism, the cell wall, the GABA shunt, and transcriptional regulators were observed in response to hypoxia. A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation. Analysis of changes in transcription factor mRNA abundance shows that hypoxia induces significant positive and negative changes that may be important for regulating the hypoxia response in this pathogenic mold. Growth in hypoxia resulted in changes in the protein levels of several glycolytic enzymes, but these changes were not always reflected by the corresponding transcriptional profiling data. However, a good correlation overall (R²= 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points. The lack of correlation between some transcript levels and their subsequent protein levels suggests another regulatory layer of the hypoxia response in <it>A. fumigatus</it>.</p> <p>Conclusions</p> <p>Taken together, our data suggest a robust cellular response that is likely regulated both at the transcriptional and post-transcriptional level in response to hypoxia by the human pathogenic mold <it>A. fumigatus</it>. As with other pathogenic fungi, the induction of glycolysis and transcriptional down-regulation of the TCA cycle and oxidative phosphorylation appear to major components of the hypoxia response in this pathogenic mold. In addition, a significant induction of the transcripts involved in ergosterol biosynthesis is consistent with previous observations in the pathogenic yeasts <it>Candida albicans </it>and <it>Cryptococcus neoformans </it>indicating conservation of this response to hypoxia in pathogenic fungi. Because ergosterol biosynthesis enzymes also require iron as a co-factor, the increase in iron uptake transcripts is consistent with an increased need for iron under hypoxia. However, unlike <it>C. albicans </it>and <it>C. neoformans</it>, the GABA shunt appears to play an important role in reducing NADH levels in response to hypoxia in <it>A. fumigatus </it>and it will be intriguing to determine whether this is critical for fungal virulence. Overall, regulatory mechanisms of the <it>A. fumigatus </it>hypoxia response appear to involve both transcriptional and post-transcriptional control of transcript and protein levels and thus provide candidate genes for future analysis of their role in hypoxia adaptation and fungal virulence.</p

Redox Proteomic Analysis Reveals Oxidative Modifications of Proteins by Increased Levels of Intracellular Reactive Oxygen Species During Hypoxia Adaptation of Aspergillus fumigatus

We thank Silke Steinbach, Till Kindel, and Michael Cyrulies for their excellent technical assistance. Work of T.K., O.K. and A.A.B was supported by the Deutsche Forschungsge-meinschaft within the Collaborative Research Center TR124 FungiNet (project A1 and Z2).The work of E.S. was supported by the International Leibniz Research School for Microbial and Biomolecular Interactions (ILRS)and by the Medical Research Council Centre for Medical Mycology at the University of Aberdeen (MR/N006364/1).We thank Matthew Blango and Falk Hillmann for the critical reading of the manuscript.Peer reviewedPostprin