Höfische Eleganz: Velázquez’ Bildnis einer Dame

Der spanische Maler Diego Rodriguez de Silva y Velázquez (1599-1660) präsentiert in dem Berliner Bildnis eine elegante, aber bis heute nicht eindeutig identifizierte Dame in entspannter Körperhaltung in Dreiviertelansicht vor einem einfarbigen, braun-beigen Hintergrund (Abb. 1). Zwar deutet sich auf den Lippen der Porträtierten ein scheues, zurückgenommenes Lächeln an, doch lässt ihr direkt auf den Betrachter gerichteter Blick aus dunklen Augen sie nichtsdestotrotz selbstbewusst erscheinen

Expression und Untersuchung von Enzymen der Pederin-Biosynthese aus einem nicht kultivierten Symbionten

Die vorliegende Arbeit beschäftigt sich mit Enzymen des ped-Genclusters aus einem bisher nicht kultivierten Symbionten. Der ped-Cluster gehört zu Typ-I-Polyketidsynthasen (PKS), in denen die Acyltrans-ferasen (AT) in trans agieren. Die PKS ist modular aufgebaut, wobei jedes Modul in Domänen untergliedert ist. Zwei Module bestehen aus Nichtribosomalen Peptid-synthetasen (NRPS). Dem Cluster wird die Herstellung von Pederin zugeschrieben, eine sehr wirksame Antitumorsubstanz, die in Käfern der Gattung Paederus und Paederidus gefunden werden kann. Die eigentliche Biosynthese von Pederin wird wahrscheinlich durch einen in den Käfern lebenden bakteriellen Symbionten durchgeführt, der ein naher Verwandter von Pseudomonas aeruginosa ist. Für genauere Hinweise auf die Funktionen einzelner Enzyme während der Pederin-Biosynthese wurden verschiedene Gene des putativen Genclusters kloniert und exprimiert. Die aufgereinigten Proteine konnten anschließend in Enzym-Assays eingesetzt werden. Hauptaspekt der Arbeit waren dabei zum einen die AT und Acylcarrierproteine (ACP) und zum anderen die O-Methyltransferasen (O-MT). Dabei sollten Substrat- und ACP-Spezifität der AT und die Regioselektivität der O-MT näher beleuchtet werden. Von den untersuchten ACP wurde PedN, codiert als separates Gen, und PedI3, eine Doppel-ACP innerhalb eines Moduls, erfolgreich als His8-Fusionsprotein exprimiert. Die Umsetzung vom apo- zum aktiven holo-ACP gelang durch die Verwendung von Svp, eine Phosphopantetheinyltransferase aus Streptomyces verticillus ATCC15003. Die Bestätigung der erfolgten Phosphopantetheinylierung erfolgte durch MALDI-TOF- bzw. ESI-FT-ICR-MS. Die beiden vorhandenen AT PedC und PedD konnten als MBP- bzw. His8-Fusionsprotein hergestellt werden. Eine Expression von PedC mit N-terminalen His8 brachte nur unlösliches Protein hervor. Zur Untersuchung der ACP-AT-Assays konnte die Verwendung von radioaktiv markierten Substraten als geeignete Methode herausgestellt werden. Dabei zeigte sich, dass PedD in der Lage ist, Malonyl-CoA zu übertragen, während es Acetyl-CoA nicht als Substrat nutzen kann. Der Transfer von Malonyl-CoA erfolgt dabei sowohl auf PedN als auch auf PedI3. Die AT weist demnach eine Substrat- jedoch keine ACP-Spezifität auf. PedC zeigte weder gegenüber Malonyl- noch Acetyl-CoA Aktivität und ist nach bisherigen Daten nicht in der Lage, das Substrat auf die ACP zu übertragen. Am Ende wurde auch die GCN5-verwandte N-Acetyltransferase (GNAT) mit dem darauffolgenden ACP kloniert und als His8-Fusionsprotein exprimiert. Bei der GNAT könnte es sich um den Überträger des Startersubstrates Acetyl-CoA handeln. Kinetische Untersuchungen des Transfers von Malonyl-CoA auf PedN durch PedD wurden begonnen. Die Durchführung dieser Untersuchung muss jedoch noch optimiert werden, um die entsprechenden Parameter kcat und Km zu ermitteln. Da Pederin vier Methoxygruppen, der ped-Cluster aber nur die drei O-MT PedA, PedE und PedO enthält, sollten diese untersucht werden. Sie wurden jeweils als MBP- und His8-Fusionsprotein erfolgreich exprimiert. Als Substratanaloga dienten Mycalamid A und B aus dem Schwamm Mycale hentscheli, die weniger O-Methylierungen als Pederin aufweisen, ihm strukturell aber sehr ähneln. Die Enzym-Assays von Fusionsprotein, Substrat und S-Adenosylmethionin (SAM) als Kofaktor wurden mittels ESI-micrOQ-TOF-MS analysiert. Dabei zeigte sich, dass PedO in der Lage ist, eine Hydroxylgruppe in Mycalamid A zu methylieren. MS/MS-Versuche stellten heraus, dass es sich um die Methylierung der OH-Gruppe an C17 oder C18 handeln muss. Die Derivatisierung des Reaktionsproduktes sowie LC-MS brachten keine spezifischeren Aussagen. Die Analyse des Reaktionsproduktes aus PedO und Mycalamid A mittels 1D- und 2D-NMR lieferte aber das Ergebnis, dass es sich um 18-O-Methylmycalamid A handeln muss. Diese durch PedO erzeugte Substanz ist nicht als Naturstoff bekannt und stellt ein neues Mitglied der Mycalamide dar. Damit wurde der erste funktionelle Beweis erbracht, dass der ped-Gencluster für die Biosynthese von Pederin verantwortlich ist. OnnD, eine O-MT aus dem onn-Gencluster (putativer Cluster der Biosynthese von Onnamid A aus Theonella swinhoei) wurde erfolgreich als MBP-Fusionsprotein exprimiert. Der entsprechende Enzym-Assay mit Pederin als Substrat zeigte keine O-Methylierung. Demnach scheint OnnD nicht für die Methoxygruppe an C13 zuständig zu sein, oder Pederin wird als Strukturanaloga nicht akzeptiert. Für zukünftige Versuche wurden die Oxidoreduktase PedB, die beiden A-Domänen, die Ketosynthase PedM und die HMGCoA-Synthase PedP als MBP- und teilweise als His8-Fusionsprotein erfolgreich exprimiert. Die beiden letztgenannten Enzyme sind wahrscheinlich an der Bildung der Exomethylengruppe an C4 in Pederin beteiligt. Die A-Domänen sind Bestandteile der NRPS und für die Auswahl und Aktivierung der jeweils einzubauenden Aminosäure erforderlich.Expression and analysis of enzymes of the pederin biosynthetic pathway from an uncultivated symbiont Presented here are the results of work carried out on the ped-cluster enzymes from an uncultivated symbiont. The ped-cluster encodes a type I polyketide synthase (PKS), in which the acyltransferases (AT) act in trans. The PKS consists of modules that are divided into domains. Two of these modules harbor non-ribosomal peptide synthetases (NRPS). Strong evidence exists that the cluster encodes the biosynthesis of pederin, a highly active antitumor compound. Pederin can be found in beetles of the genera Paederus and Paederidus, but biosynthesis of pederin is performed by a bacterial symbiont that lives in the beetle and that has a close relationship to Pseudomonas aeruginosa. In order to gain further insights into the function of distinct enzymes during the biosynthesis of pederin, different genes of the putative gene cluster were cloned and expressed. The purified enzymes were then used for enzyme assays. An important aspect of the work was to look at the AT and acyl carrier proteins (ACP) as well as the O-methyltransferases (O-MT). Investigations into substrate- and ACP-specifity of the AT and regioselectivity of the O-MT was carried out. Successful expression as His8-fusion protein in the case of the tested ACP was carried out for PedN, encoded as a separate gene, and PedI3, a didomain-ACP within a module of the PKS. Use of Svp, a phosphopantetheinyltransferase from Streptomyces verticillus ATCC15003, leads to conversion of apo-ACP, forming an active holo-ACP. Confirmation of the successful phosphopantetheinylation was ascertained using MALDI-TOF- and ESI-FT-ICR-MS. The two available AT PedC und PedD were produced as maltose binding- or His8-fusion proteins. Expression of PedC with N-terminal histidine tag resulted only in insoluble protein. For analysis of the ACP-AT-assays the use of radioactive labeled substrates was found to be the favourable method. This method showed that PedD is able to use malonyl-CoA as a substrate, but not acetyl-CoA. PedD transfers malonyl-CoA to PedN as well as to PedI3. The AT PedD showed substrate, but not ACP-specifity. PedC had no affinity for malonyl- or acetyl-CoA. Preliminary data suggest that PedC is not able to transfer any substrate to the ACP. The GCN5-related N-acetyltransferase (GNAT) with the neighbouring ACP was also cloned and expressed as His8-fusion protein. GNAT is probably responsible for the transfer of the initiation substrate, acetyl-CoA. Kinetic experiments for the transfer of malonyl-CoA to PedN by PedD have been started, but the procedure must still be optimized in order to obtain the appropriate parameters kcat and Km. Because pederin has four methoxygroups, but the ped-cluster only contains three O-MT, these three enzymes (PedA, PedE and PedO) were examined. Each of them was successfully expressed as maltose binding- and as His8-fusion proteins. Mycalamide A and B from the sponge Mycale hentscheli were used as substrate analogues. They have less O-methylations, but their structure is very close to that of pederin. The enzyme-assays of the fusion proteins were carried out with substrate and the cofactor S-adenosylmethionine (SAM). These were analysed by ESI-micrOQ-TOF-MS. This showed that PedO is able to methylate one hydroxyl group of mycalamide A. MS/MS-experiments revealed that the methylation occurs at the C17 or C18 OH-group. Derivatisation of the reaction product or LC-MS did not provide more specific conclusions. Analysis of the product of PedO and mycalamide A using 1D- and 2D-NMR showed that 18-O-methylmycalamide A was formed. This substance is not a known natural product and is a new member of the mycalamides. These results represent the first functional proof that ped-gene cluster encodes for the biosynthesis of pederin. The O-MT OnnD from the onn-cluster (putative gene cluster for biosynthesis of onnamide A from Theonella swinhoei) was successfully expressed as maltose binding fusion protein. The enzyme assay with pederin as the substrate showed no O-methylation. It seems that OnnD is either not responsible for the methylation of OH at C13, or it does not accept pederin as structural analogue. For future experiments, the oxidoreductase PedB, the two A-domains, the ketosynthase PedM and the HMGCoA-synthase PedP have been successfully expressed as maltose binding- and partly as His8-fusion proteins. The latter two enzymes are likely involved in the formation of the exomethylene group at C4 of pederin. The A-domains, which are part of the NRPS, are responsible for the choice and activation of the amino acids that are built in

Ulcerating Ileocolitis in Severe Amatoxin Poisoning

Amatoxin poisoning is still associated with a great potential for complications and a high mortality. While the occurrence of acute gastroenteritis within the first 24 hours after amatoxin ingestion is well described, only very few descriptions of late gastrointestinal complications of amatoxin poisoning exist worldwide. We present the case of a 57-year-old female patient with severe amatoxin poisoning causing fulminant but reversible hepatic failure that on day 8 after mushroom ingestion developed severe abdominal pain and watery diarrhea. Ulcerating ileocolitis was identified by computed tomography identifying a thickening of the bowel wall of the entire ileum and biopsies taken from the ileum and large bowel revealing distinct ileitis and proximally accentuated colitis. The absence of discernible alternative etiologies such as infectious agents makes a causal relationship between the ulcerating ileocolitis and the amatoxin poisoning likely. Diarrhea and varying abdominal pain persisted over several weeks and clinical follow-up after six months showed a completely symptom-free patient. The case presented highlights the importance to consider the possibility of rare complications of Amanita intoxication in order to be able to respond to them early and adequately

Highly efficient concentration of lenti- and retroviral vector preparations by membrane adsorbers and ultrafiltration

<p>Abstract</p> <p>Background</p> <p>Lentiviral vectors (LVs) can efficiently transduce a broad spectrum of cells and tissues, including dividing and non-dividing cells. So far the most widely used method for concentration of lentiviral particles is ultracentrifugation (UC).</p> <p>An important feature of vectors derived from lentiviruses and prototypic gamma-retroviruses is that the host range can be altered by pseudotypisation. The most commonly used envelope protein for pseudotyping is the glycoprotein of the Vesicular Stomatitis Virus (VSV.G), which is also essential for successful concentration using UC.</p> <p>Results</p> <p>Here, we describe a purification method that is based on membrane adsorbers (MAs). Viral particles are efficiently retained by the anionic exchange MAs and can be eluted with a high-salt buffer. Buffer exchange and concentration is then performed by utilizing ultrafiltration (UF) units of distinct molecular weight cut off (MWCO). With this combined approach similar biological titers as UC can be achieved (2 to 5 × 10⁹infectious particles (IP)/ml). Lentiviral particles from small starting volumes (e.g. 40 ml) as well as large volumes (up to 1,000 ml) cell culture supernatant (SN) can be purified. Apart from LVs, vectors derived from oncoretroviruses can be efficiently concentrated as well. Importantly, the use of the system is not confined to VSV.G pseudotyped lenti- and retroviral particles and other pseudotypes can also be purified.</p> <p>Conclusions</p> <p>Taken together the method presented here offers an efficient alternative for the concentration of lenti- as well as retroviral vectors with different pseudotypes that needs no expensive equipment, is easy to handle and can be used to purify large quantities of viral vectors within a short time.</p

Low Magnetic Field Regime of a Gate-Defined Constriction in High-Mobility Graphene

We report on the evolution of the coherent electronic transport through a gate-defined constriction in a high-mobility graphene device from ballistic transport to quantum Hall regime upon increasing the magnetic field. At low field, the conductance exhibits Fabry-P\'erot resonances resulting from the npn cavities formed beneath the top-gated regions. Above a critical field $B^*$ corresponding to the cyclotron radius equal to the npn cavity length, Fabry-P\'erot resonances vanish and snake trajectories are guided through the constriction with a characteristic set of conductance oscillations. Increasing further the magnetic field allows us to probe the Landau level spectrum in the constriction, with distortions due to the combination of confinement and de-confinement of Landau levels in a saddle potential. These observations are confirmed by numerical calculations

Expression of MAGE-C1/CT7 and selected cancer/testis antigens in ovarian borderline tumours and primary and recurrent ovarian carcinomas

MAGE-C1/CT7, NY-ESO-1, GAGE and MAGE-A4 are members of the cancer/testis (CT) antigen family, which have been proposed as potential targets for cancer immunotherapy. To determine the prevalence and biologic relevance of the novel CT antigen MAGE-C1/CT7 and other antigens, 36 ovarian borderline tumours (BTs), 230 primary ovarian carcinomas (OCs) and 80 recurrent OCs were immunohistochemically analysed using the monoclonal antibodies CT7-33 (MAGE-C1/CT7), E978 (NY-ESO-1), clone 26 (GAGE) and 57B (MAGE-A4). Positivity of at least one CT antigen was present in 39.5% (81/205) of primary OC and in 50% (26/52) of all recurrences. Expression of the novel CT antigen MAGE-C1/CT7 was most commonly seen with positivity in 24.5% of primary and 35.1% of recurrent OC. MAGE-A4, GAGE and NY-ESO-1 expressions were seen in 22.7, 13.9 and 7.1% of primary and 22.6, 17.5 and 8.9% of recurrent OC, respectively. Analysis of histological subtypes (serous, endometrioid, clear cell, mucinous and transitional) exhibited variable expression with negativity in all mucinous OC. High-grade serous OC revealed CT antigen expression in 5.6 to 28% with MAGE-C1/CT7 being the most frequent, but without correlation with stage or overall survival. MAGE-C1/CT7 expression and coexpression of CT antigens were significantly correlated with grade of endometrioid OC. None of the BT showed CT antigen expression. No significant correlation was seen with stage, overall survival or response to chemotherapy. In summary, CT antigens are expressed in a certain subset of OC with no expression in BT or OC of mucinous histology. These findings may have implications for the design of polyvalent vaccination strategies for ovarian carcinoma

Präklinische Polytraumaversorgung

Tscherne was the first to define the term polytrauma in 1966 as “multiple injuries to different regions of the body sustained simultaneously, with at least one injury or the combination of these injuries being life-threatening”. This definition highlights the essential pathophysiological paradigm of polytrauma, with the life-threating characteristics resulting from injuries to multiple organ systems. The treatment of polytrauma patients begins at the scene of the accident. Important life-saving initial interventions can already be carried out on site through targeted measures and expertise of the emergency medical service team, thus improving patient survival. The advanced trauma life support/prehospital trauma life support (ATLS/PHTLS) concept is the worldwide gold standard. As prehospital treatment of severely injured patients is not routine for most emergency teams, concepts and emergency interventions must be regularly trained. This is the prerequisite for safe and effective emergency treatment in this time-critical situation