Charakterisierung des neu entwickelten monoklonalen Antikörpers UMB-3 gegen den -Opioidrezeptor

Die Lokalisation des µ-Opioidrezeptors (MOR) im Gehirn von Nagern konnte in der Vergangenheit bereits mit Hilfe polyklonaler Antikörper untersucht werden. Schwierigkeiten bereitete die Herstellung von Antikörpern für die Immunpräzipitation und das Immunoblotting von MOR aus nativem Gewebe. Ursächlich hierfür ist die geringe Menge an MOR in vivo. Die biochemische Analyse von MOR-Signalwegen ist damit erschwert. Zudem sind die bisherigen immunhistochemischen Untersuchungen zur Lokalisation des MOR auf Gefrierschnitte beschränkt gewesen. Dies liegt an der begrenzten kommerziellen Verfügbarkeit von Anti-MOR-Antikörpern, die in der Lage sind, den labilen MOR in in Paraffin eingebetteten Geweben nachzuweisen. Ziel der vorliegenden Arbeit war die Charakterisierung des neu entwickelten monoklonalen Anti-MOR-Antikörpers UMB-3. Hierzu wurden Western-Blot-Analysen nach Rezeptorisolierung aus transfizierten MOR-Zellen und aus Hirngewebe von MOR(+/+)- und MOR(-/-)-Mäusen, immunzytochemische Studien an transfizierten MOR-Zellen sowie immunhistochemische Untersuchungen an Paraffinschnitten von gesunden Nagerorganen und von humanen Tumorgeweben durchgeführt. Die Spezifität des UMB-3 konnte durch folgende Ergebnisse demonstriert werden: 1) Die Detektion einer breiten Bande im Bereich von 70-80kDa bei den MOR-Zellen und in den Immunpräzipitaten aus MOR(+/+)-, nicht jedoch aus MOR(-/-)-Hirnen. 2) Eine membranständige Färbung von unstimulierten MOR-Zellen sowie die Translokation des Fluoreszenzsignals nach der Behandlung mit einem MOR-Agonisten. 3) Die Markierung von Nervenzellkörpern und -fortsätzen in MOR-exprimierenden Regionen des Nervensystems. 4) Die Abwesenheit einer solchen Färbung in den MOR-Knockout-Mäusen bzw. nach der Peptidneutralisation. Zusammenfassend konnte gezeigt werden, dass sich der UMB-3 sowohl für die Immunpräzipitation und das Immunoblotting des MOR aus nativem Gewebe als auch für dessen Detektion in Paraffinschnitten von Geweben aus Nagern und Menschen eignet

Spatial and temporal dynamics of suspended particles and E. coli in a complex surface-water and karst groundwater system as a basis for an adapted water protection scheme, northern Vietnam [复杂地表水和喀斯特地下水系统中悬浮颗粒和大肠杆菌的时空动态演变–越南北部调整水资源保护方案的基础] [Dynamique spatiale et temporelle des particules en suspension et d’E. Coli dans un système complexe d’eaux de surface et d’eaux souterraines karstiques comme base d’un programme adapté à la protection de l’eau, nord du Vietnam] [Dinâmica espacial e temporal de partículas suspensas e E. coli em um sistema complexo de águas superficial e subterrânea cársticas como base para um esquema adaptado de proteçã o de água, norte do Vietnã] [Dinámica espacial y temporal de partículas en suspensión y E. coli en un complejo sistema de aguas superficiales y subterráneas kársticas como base para un plan adaptativo de protección del agua, norte de Vietnam]

Karst aquifers in subtropical regions are characterized by high variability of water availability and quality due to changes associated with rainy and dry seasons. An additional challenge for water management is the combination of surface-water and karst groundwater systems since high spatiotemporal dynamics cause high variability of water quality. In these cases, adapted protection strategies are required. In this study, a protection approach for the catchment of a river-water diversion point in a rural area in northern Vietnam is developed. The variability of water quality was evaluated by rainy and dry season synoptic surveys of suspended particles and microbial contamination at 49 sites and time series at three sets of paired sites under constant hydraulic conditions. The anthropogenic land-use activities in the catchment were mapped to identify potential contamination sources and to highlight the challenging combination of surface-water and karst groundwater management. The analyzed data indicate differences in water quality between the dry and rainy seasons and a higher influence on water quality from land use than from hydrologic conditions. Furthermore, the results suggest a high risk of contamination resulting from residential areas, agriculture, and livestock farming, and reveal the necessity of implementation of appropriate measures such as restricted farming and the hook-up of buildings to municipal sewage disposal. Finally, the data show that water quality can be improved by adjusting water withdrawals by the time of day. The applied methods can be transferred to other surface-water and karst groundwater systems in similar subtropical environments

Better Together: Engaging Stakeholders in Learning and Leadership to Guide Foundation Resources Toward Adaptive Systems Change

In 2014, the Kansas Health Foundation brought together a group of knowledgeable stakeholders from a multitude of specialties to focus on reducing tobacco use specifically among Kansans with mental illness. Over 15 months, the group and the foundation worked to learn deeply about the issue and inform action that could be taken on individual, organizational, and systemic levels. The wealth of knowledge and experience brought by each participant to the discussion and learning about this complex issue, together from a range of perspectives, resulted in a more productive dialogue. The model proved very effective, as evidenced by the group’s success in achieving a number of policy, system, and environmental changes — including expanding cessation benefits available under Medicaid in Kansas — and could be replicated by any foundation. The foundation continues to work collaboratively on this issue and discover more about what is effective in reducing tobacco use. What it learned alongside its community partners has powerfully informed the foundation’s approach to this work and has resulted in meaningful change, at multiple levels, in the behavioral health system

Studies on the in vitro and in vivo metabolism of the synthetic opioids U-51754, U-47931E, and methoxyacetylfentanyl using hyphenated high-resolution mass spectrometry

New Synthetic Opioids (NSOs) are one class of New Psychoactive Substances (NPS) enjoying increasing popularity in Europe. Data on their toxicological or metabolic properties have not yet been published for most of them. In this context, the metabolic fate of three NSOs, namely, trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzenacetamide (U-51754), trans-4-bromo-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide (U-47931E), and 2-methoxy-N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl] acetamide (methoxyacetylfentanyl), was elucidated by liquid chromatography high-resolution mass spectrometry after pooled human S9 fraction (phS9) incubations and in rat urine after oral administration. The following major reactions were observed: demethylation of the amine moiety for U-51754 and U-47931E, N-hydroxylation of the hexyl ring, and combinations thereof. N-dealkylation, O-demethylation, and hydroxylation at the alkyl part for methoxyacetylfentanyl. Except for U-47931E, parent compounds could only be found in trace amounts in rat urine. Therefore, urinary markers should preferably be metabolites, namely, the N-demethyl-hydroxy and the hydroxy metabolite for U-51754, the N-demethylated metabolite for U-47931E, and the N-dealkylated metabolite as well as the O-demethylated one for methoxyacetylfentanyl. In general, metabolite formation was comparable in vitro and in vivo, but fewer metabolites, particularly those after multiple reaction steps and phase II conjugates, were found in phS9. These results were consistent with those of comparable compounds obtained from human liver microsomes, human hepatocytes, and/or human case studies

Open Educational Resources (OER) in der wissenschaftlichen Weiterbildung

Während sich die meisten Arbeiten über (freie) digitale Bildungsressourcen im Bereich der allgemeinen Hochschulbildung bewegen, nähert sich dieser Beitrag dem Thema aus der Perspektive der wissenschaftlichen Weiterbildung. Zunächst werden freie Bildungsmaterialien (OER) entlang der verschiedenen Creative Commons Lizenzen gegenüber digitalen Bildungsressourcen allgemein begriffl ich abgegrenzt. Eine Herausforderung für Weiterbildungsanbieter bzw. für das Weiterbildungspersonal besteht zudem in der Auswahl qualitativ hochwertiger Materialien. Hierzu wird auf die verschiedenen Qualitätsdimensionen für digitale Lernmaterialien und auch auf die Schwierigkeiten bei der praktischen Implementation der Qualitätssicherung eingegangen. Weiterbildungseinrichtungen bewegen sich mit ihren kostenpfl ichtigen Angeboten in einem Markt, in dem der Besitz bzw. das Copyright an Lernmaterialien, deren Entwicklung oft sehr kostenintensiv ist, als ein Wettbewerbsvorteil gesehen wird. Es besteht hier die Tendenz, Lernmaterialien eher nicht als OER zu veröffentlichen. Vor dem Hintergrund dieses Spannungsfeldes wird im Rahmen einer Einzelfallstudie ein Projekt vorgestellt, in dem OER in der wissenschaftlichen Weiterbildung für das in Aufbau befi ndliche OER-Portal Niedersachsen erstellt werden

The BDSF quorum sensing receptor RpfR regulates Bep exopolysaccharide synthesis in Burkholderia cenocepacia via interaction with the transcriptional regulator BerB

The polysaccharide Bep is essential for in vitro biofilm formation of the opportunistic pathogen Burkholderia cenocepacia. We found that the Burkholderia diffusible signaling factor (BDSF) quorum sensing receptor RpfR is a negative regulator of the bep gene cluster in B. cenocepacia. An rpfR mutant formed wrinkled colonies, whereas additional mutations in the bep genes or known bep regulators like berA and berB restored the wild-type smooth colony morphology. We found that there is a good correlation between intracellular c-di-GMP levels and bep expression when the c-di-GMP level is increased or decreased through ectopic expression of a diguanylate cyclase or a c-di-GMP phosphodiesterase, respectively. However, when the intracellular c-di-GMP level is changed by site directed mutagenesis of the EAL or GGDEF domain of RpfR there is no correlation between intracellular c-di-GMP levels and bep expression. Except for rpfR, deletion mutants of all 25 c-di-GMP phosphodiesterase and diguanylate cyclase genes encoded by B. cenocepacia showed no change to berA and bep gene expression. Moreover, bacterial two-hybrid assays provided evidence that RpfR and BerB physically interact and give specificity to the regulation of the bep genes. We suggest a model where RpfR binds BerB at low c-di-GMP levels to sequester this RpoN-dependent activator to an RpfR/RpfF complex. If the c-di-GMP levels rise, possibly by the enzymatic action of RpfR, BerB binds c-di-GMP and is released from the RpfR/RpfF complex and associates with RpoN to activate transcription of berA, and the BerA protein subsequently activates transcription of the bep genes

Kinetics of Ethylene and Ethylene Oxide in Subcellular Fractions of Lungs and Livers of Male B6C3F1 Mice and Male Fischer 344 Rats and of Human Livers

Ethylene (ET) is metabolized in mammals to the carcinogenic ethylene oxide (EO). Although both gases are of high industrial relevance, only limited data exist on the toxicokinetics of ET in mice and of EO in humans. Metabolism of ET is related to cytochrome P450-dependent mono-oxygenase (CYP) and of EO to epoxide hydrolase (EH) and glutathione S-transferase (GST). Kinetics of ET metabolism to EO and of elimination of EO were investigated in headspace vessels containing incubations of subcellular fractions of mouse, rat, or human liver or of mouse or rat lung. CYP-associated metabolism of ET and GST-related metabolism of EO were found in microsomes and cytosol, respectively, of each species. EH-related metabolism of EO was not detectable in hepatic microsomes of rats and mice but obeyed saturation kinetics in hepatic microsomes of humans. In ET-exposed liver microsomes, metabolism of ET to EO followed Michaelis-Menten-like kinetics. Mean values of Vmax [nmol/(min·mg protein)] and of the apparent Michaelis constant (Km [mmol/l ET in microsomal suspension]) were 0.567 and 0.0093 (mouse), 0.401 and 0.031 (rat), and 0.219 and 0.013 (human). In lung microsomes, Vmax values were 0.073 (mouse) and 0.055 (rat). During ET exposure, the rate of EO production decreased rapidly. By modeling a suicide inhibition mechanism, rate constants for CYP-mediated catalysis and CYP inactivation were estimated. In liver cytosol, mean GST activities to EO expressed as Vmax/Km [μl/(min·mg protein)] were 27.90 (mouse), 5.30 (rat), and 1.14 (human). The parameters are most relevant for reducing uncertainties in the risk assessment of ET and EO

Cytosolic monothiol glutaredoxins function in intracellular iron sensing and trafficking via their bound iron-sulfur cluster

Iron is an essential nutrient for cells. It is unknown how iron, after its import into the cytosol, is specifically delivered to iron-dependent processes in various cellular compartments. Here, we identify an essential function of the conserved cytosolic monothiol glutaredoxins Grx3 and Grx4 in intracellular iron trafficking and sensing. Depletion of Grx3/4 specifically impaired all iron-requiring reactions in the cytosol, mitochondria, and nucleus, including the synthesis of Fe/S clusters, heme, and di-iron centers. These defects were caused by impairment of iron insertion into proteins and iron transfer to mitochondria, indicating that intracellular iron is not bioavailable, despite highly elevated cytosolic levels. The crucial task of Grx3/4 is mediated by a bridging, glutathione-containing Fe/S center that functions both as an iron sensor and in intracellular iron delivery. Collectively, our study uncovers an important role of monothiol glutaredoxins in cellular iron metabolism, with a surprising connection to cellular redox and sulfur metabolisms

Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells

Background: Mesenchymal stromal cells isolated from bone marrow (MSC) represent an attractive source of adult stem cells for regenerative medicine. However, thorough research is required into their clinical application safety issues concerning a risk of potential neoplastic degeneration in a process of MSC propagation in cell culture for therapeutic applications. Expansion protocols could preselect MSC with elevated levels of growth-promoting transcription factors with oncogenic potential, such as c-MYC. We addressed the question whether c-MYC expression affects the growth and differentiation potential of human MSC upon extensive passaging in cell culture and assessed a risk of tumorigenic transformation caused by MSC overexpressing c-MYC in vivo. Methods: MSC were subjected to retroviral transduction to induce expression of c-MYC, or GFP, as a control. Cells were expanded, and effects of c-MYC overexpression on osteogenesis, adipogenesis, and chondrogenesis were monitored. Ectopic bone formation properties were tested in SCID mice. A potential risk of tumorigenesis imposed by MSC with c-MYC overexpression was evaluated. Results: C-MYC levels accumulated during ex vivo passaging, and overexpression enabled the transformed MSC to significantly overgrow competing control cells in culture. C-MYC-MSC acquired enhanced biological functions of c-MYC: its increased DNA-binding activity, elevated expression of the c-MYC-binding partner MAX, and induction of antagonists P19ARF/P16INK4A. Overexpression of c-MYC stimulated MSC proliferation and reduced osteogenic, adipogenic, and chondrogenic differentiation. Surprisingly, c-MYC overexpression also caused an increased COL10A1/COL2A1 expression ratio upon chondrogenesis, suggesting a role in hypertrophic degeneration. However, the in vivo ectopic bone formation ability of c-MYC-transduced MSC remained comparable to control GFP-MSC. There was no indication of tumor growth in any tissue after transplantation of c-MYC-MSC in mice. Conclusions: C-MYC expression promoted high proliferation rates of MSC, attenuated but not abrogated their differentiation capacity, and did not immediately lead to tumor formation in the tested in vivo mouse model. However, upregulation of MYC antagonists P19ARF/P16INK4A promoting apoptosis and senescence, as well as an observed shift towards a hypertrophic collagen phenotype and cartilage degeneration, point to lack of safety for clinical application of MSC that were manipulated to overexpress c-MYC for their better expansion