Diagnostische Bedeutung von Telomerase-Aktivität in Perikardergüssen

Die Erkennung von malignen Zellen in Perikardergüssen ist ein bleibendes Problem in der klinischen Diagnostik. Als Goldstandard für die Detektion von Tumorzellen in Ergüssen gilt zur Zeit die Zytologie. Trotzdem werden bei dieser Untersuchung regelmäßig maligne Zellen übersehen. Ziel der vorliegenden Arbeit war es daher, den Nutzen der Telomerase-Aktivitätsmessung für die Begutachtung von Perikardergüssen herauszufinden, um möglicherweise neben der Zytologie ein weiteres Entscheidungskriterium zur Beurteilung der Malignität von Ergüssen zu erhalten. In der vorliegenden Arbeit wurden 31 zytologisch kontrollierte Perikardergüsse auf erhöhte Telomerase-Aktivität mit Hilfe eines konventionellen und eines fluoreszenz-basierten sog. TRAP-Assays (Telomeric Repeat Amplification Protocol) untersucht. Die Ergebnisse des konventionellen TRAP-Assays zeigen eine hohe Korrelation zwischen dem Telomerase-Aktivitätsnachweis und dem zytopathologisch bestätigten Nachweis von malignen Zellen: es konnte eine Sensitivität von 100 % bei einer Spezifität von 91,7 % erreicht werden. Da niedrige Telomerase-Aktivität auch in Stammzellen oder aktivierten Entzündungszellen vorkommt und möglicherweise falsch-positive Ergebnisse provoziert, wurde im weiteren Verlauf der Arbeit versucht, die Aktivität dieses Enzyms zu quantifizieren. Durch die Quantifizierung sollten Rückschlüsse über die Herkunft der Telomerase-Aktivität gewonnen werden. Dazu wurde der fluoreszenz-basierte F-TRAP-Assay etabliert und anhand eines Standards einer telomerase-positiven kleinzelligen Bronchialkarzinomlinie (NCI-H69) kalibriert. Bei den Versuchen wurden nur geringfügige Mess-Schwankungen der Telomerase-Aktivität innerhalb der Verdünnungsreihen registriert. Dies bedeutet eine wenn auch eingeschränkte Quantifizierbarkeit der Telomerase-Aktivität. Daher wurden weitere Untersuchungen vorgenommen, um die Ursache für diese Abweichungen zu ermitteln. Es stellte sich heraus, dass die Schwankungen durch die Elongation und nicht durch die PCR-Amplifikation verursacht werden. Die Elongationsphase stellt damit das Kernproblem bei der Quantifizierung von Telomerase-Aktivität dar. Des Weiteren wurde die Hintergrundaktivität im Perikarderguss anhand von CD34+-Zellen und peripheren mononukleären Zellen aus peripherem Blut überprüft. Diese Untersuchung erfolgte zu dem Zweck, die mögliche Kontamination eines Perikardergusses durch proliferierende Entzündungszellen zu simulieren und gleichzeitig einen „Cut-off“-Wert für Hintergrundaktivität zu generieren. In beiden Zellarten wurde lediglich ein niedriger Level an Telomerase-Aktivität registriert. Aufgrund der Diskrepanz zwischen den Aktivitätsspiegeln benigner Entzündungszellen im Vergleich zu malignen Tumorzellen konnten Rückschlüsse auf die Herkunft der Telomerase-Aktivität gewonnen werden. Obwohl eine absolute Quantifizierung von Telomerase-Aktivität nicht möglich war, konnte durch die Einführung des „Cut-off“-Wertes die Spezifität durch den F-TRAP-Assay auf 95,8 % gesteigert werden ohne einen Verlust an Sensitivität hinnehmen zu müssen. Die hohe Sensitivität beider Assays weist auf die besondere Eignung von Perikardzytomaterial für Telomerase-Aktivitätsmessungen hin. Hierzu hat möglicherweise auch eine gute Qualität der verwendeten Proteinextrakte mit einem vermutlich geringen Anteil an Proteinasen und RNAsen beigetragen. Die Ergebnisse beider TRAP-Assays zeigen, dass die Bestimmung der Telomerase-Aktivität ein reliabler Indikator für das Vorhandensein von Tumorzellen in Perikardergüssen ist und damit eine sinnvolle Ergänzung zur zytologischen Untersuchung darstellt. Insbesondere in Zweifelsfällen könnte die Telomerase-Aktivitätsmessung aufschlussreiche Zusatzinformationen bieten. Die Untersuchung wird derzeit aufgrund des methodischen Aufwands jedoch noch Speziallabors vorbehalten sein

Nanoporous Gold: From Structure Evolution to Functional Properties in Catalysis and Electrochemistry

Nanoporous gold (NPG) is characterized by a bicontinuous network of nanometer-sized metallic struts and interconnected pores formed spontaneously by oxidative dissolution of the less noble element from gold alloys. The resulting material exhibits decent catalytic activity for low-temperature, aerobic total as well as partial oxidation reactions, the oxidative coupling of methanol to methyl formate being the prototypical example. This review not only provides a critical discussion of ways to tune the morphology and composition of this material and its implication for catalysis and electrocatalysis, but will also exemplarily review the current mechanistic understanding of the partial oxidation of methanol using information from quantum chemical studies, model studies on single-crystal surfaces, gas phase catalysis, aerobic liquid phase oxidation, and electrocatalysis. In this respect, a particular focus will be on mechanistic aspects not well understood, yet. Apart from the mechanistic aspects of catalysis, best practice examples with respect to material preparation and characterization will be discussed. These can improve the reproducibility of the materials property such as the catalytic activity and selectivity as well as the scope of reactions being identified as the main challenges for a broader application of NPG in target-oriented organic synthesis

Hyperpolarization-Activated Cyclic Nucleotide-Gated Non-selective (HCN) Ion Channels Regulate Human and Murine Urinary Bladder Contractility

Purpose: Hyperpolarization-activated cyclic nucleotide gated non-selective (HCN) channels have been demonstrated in the urinary bladder in various species. Since they play a major role in governing rhythmic activity in pacemaker cells like in the sinoatrial node, we explored the role of these channels in human and murine detrusor smooth muscle.Methods: In an organ bath, human and murine detrusor smooth muscle specimens were challenged with the HCN channel blocker ZD7288. In human tissue derived from macroscopically tumor-free cancer resections, the urothelium was removed. In addition, HCN1-deficient mice were used to identify the contribution of this particular isoform. Expression of HCN channels in the urinary bladder was analyzed using histological and ultrastructural analyses as well as quantitative reverse transcriptase polymerase chain reaction (RT-PCR).Results: We found that the HCN channel blocker ZD7288 (50 μM) both induced tonic contractions and increased phasic contraction amplitudes in human and murine detrusor specimens. While these responses were not sensitive to tetrodotoxin, they were significantly reduced by the gap junction inhibitor 18β-glycyrrhetic acid suggesting that HCN channels are located within the gap junction-interconnected smooth muscle cell network rather than on efferent nerve fibers. Immunohistochemistry suggested HCN channel expression on smooth muscle tissue, and immunoelectron microscopy confirmed the scattered presence of HCN2 on smooth muscle cell membranes. HCN channels seem to be down-regulated with aging, which is paralleled by an increasing effect of ZD7288 in aging detrusor tissue. Importantly, the anticonvulsant and HCN channel activator lamotrigine relaxed the detrusor which could be reversed by ZD7288.Conclusion: These findings demonstrate that HCN channels are functionally present and localized on smooth muscle cells of the urinary bladder. Given the age-dependent decline of these channels in humans, activation of HCN channels by compounds such as lamotrigine opens up the opportunity to combat detrusor hyperactivity in the elderly by drugs already approved for epilepsy

ALD Functionalized Nanoporous Gold: Thermal Stability, Mechanical Properties, and Catalytic Activity

Nanoporous metals have many technologically promising applications but their tendency to coarsen limits their long-term stability and excludes high temperature applications. Here, we demonstrate that atomic layer deposition (ALD) can be used to stabilize and functionalize nanoporous metals. Specifically, we studied the effect of nanometer-thick alumina and titania ALD films on thermal stability, mechanical properties, and catalytic activity of nanoporous gold (np-Au). Our results demonstrate that even only one-nm-thick oxide films can stabilize the nanoscale morphology of np-Au up to 1000 C, while simultaneously making the material stronger and stiffer. The catalytic activity of np-Au can be drastically increased by TiO{sub 2} ALD coatings. Our results open the door to high temperature sensor, actuator, and catalysis applications and functionalized electrodes for energy storage and harvesting applications

Einführung in die Grundlagen der Pharmakovigilanz (Teil III): Risikobewertungsverfahren in der Europäischen Union

Nachdem in den ersten beiden Beiträgen zur Einführung in die Pharmakovigilanz die Begriffsbestimmung, Klassifizierung und Erfassung von unerwünschten Arzneimittelwirkungen(UAW) beschrieben wurden, werden im Folgenden die Verfahren zur Bewertung von Arzneimittelrisiken und die Umsetzung der daraus gewonnenen Erkenntnisse dargestellt. Die meisten Entscheidungen werden auf europäischer Ebene getroffen, rein nationale Entscheidungsspielräume sind für viele Arzneimittel nur noch sehr begrenzt vorhanden

Our contribution to the Faraday Discussion on Gold Nanoporous gold: a new gold catalyst with tunable properties

Abstract Nanoporous gold (np-Au) represents a novel nanostructured bulk material with very interesting 10 perspectives in heterogeneous catalysis. Its monolithic porous structure and the absence of a support or other stabilizing agents opens up unprecedented possibilities to tune structure and surface chemistry in order to adapt the material to specific catalytic applications. We investigated three of these tuning options in more detail: change of the porosity by annealing, increase of activity by the deposition of oxides and change of activity and selectivity by bimetallic effects. As an example for the latter case, the effect of Ag impurities will be discussed. The presence and concentration of Ag can be correlated to the availability of active oxygen. While for the oxidation of CO the activity of the catalyst can be significantly enhanced when increasing the content of Ag, we show for the oxidation of methanol that the selectivity is shifted from partial to total oxidation. In a second set of experiments, two different metal-oxides were deposited on np-Au, praseodymia and titania. In both 20 cases, the surface chemistry changed significantly. The activity of the catalyst for oxidation of CO was increased by up to one order of magnitude after modification. Finally, we used adsorbate controlled coarsening to tune the structure of np-Au. In this way, even gradients in the pore-and ligament size could be induced, taking advantage of mass transport phenomena

Surface Chemistry in Nanoscale Materials

Although surfaces or, more precisely, the surface atomic and electronic structure, determine the way materials interact with their environment, the influence of surface chemistry on the bulk of the material is generally considered to be small. However, in the case of high surface area materials such as nanoporous solids, surface properties can start to dominate the overall material behavior. This allows one to create new materials with physical and chemical properties that are no longer determined by the bulk material, but by their nanoscale architectures. Here, we discuss several examples, ranging from nanoporous gold to surface engineered carbon aerogels that demonstrate the tuneability of nanoporous solids for sustainable energy applications

Aerobic Methanol Oxidation over Unsupported Nanoporous Gold: The Influence of an Added Base

We studied the aerobic oxidation of methanol over nanoporous gold catalysts under neutral and alkaline conditions. We find that under neutral conditions the catalyst has an activation period of about 10 h while upon addition of a base the catalyst becomes active right away. After this activation period, however, the activity of the catalyst is in both cases similar. Moreover, the selectivity was not affected by the base. We tested different bases and found the largest effect when adding OH−. The cation, however, does not play a role. We conclude that it is OH−, which is impacting the reaction and propose a mechanism for the suppression of the activation period. While the catalytic cycle, i.e., the reaction of methanol on the catalyst surface seems unaffected, the transient adsorption of OH− onto the surface can facilitate the activation of molecular oxygen by donating electrons to the surface. Due to the intermediate formation of oxidic Ag species, an effective segregation of surface-near Ag can be induced, which increases the abundance of Ag being essential for the activation of oxygen at the surface. In this way, a more efficient pathway for the generation of active oxygen is opened, allowing the reaction to set in faster

Heterogeneity of the prtC gene of Porphyromonas gingivalis

In this study, the nucleotide sequences of the prtC genes of six clinical Porphyromonas gingivalis isolates obtained from patients with periodontitis and from reference strain 53977 were determined. All analyzed genes were heterogeneous in their nucleotide composition and differed in up to 13 nucleotides. Moreover, substantial differences were found in comparison to prtC of reference strain 53977. The prtC genes of 45 Porphyromonas gingivalis isolates were amplified by polymerase chain reaction (PCR) and the PCR products were also digested with restriction endonucleases Tsp509I, NlaIII and DraII (PCR-restriction fragment-length polymorphism). Nine different restriction pattern combinations were observed, with four being most frequent (28.9%, 26.7%, 17.8% and 11.1%). The data presented here demonstrates that prtC genes are heterogeneous in their nucleotide sequence and therefore may be used as a target for molecular epidemiological studies. The observed heterogeneity of prtC genes may be a result of microevolution processes.link_to_subscribed_fulltex