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- ItemQuantification of osseointegration of plasma-polymer coated titanium alloyed implants by means of microcomputed tomography versus histomorphometry(New York [u.a.] : Hindawi, 2015) Gabler, Carolin; Zietz, Carmen; Bieck, Richard; Göhler, Rebecca; Lindner, Tobias; Haenle, Maximilian; Finke, Birgit; Meichsner, JĂ¼rgen; Testrich, Holger; Nowottnick, Mathias; Frerich, Bernhard; Bader, RainerA common method to derive both qualitative and quantitative data to evaluate osseointegration of implants is histomorphometry. The present study describes a new image reconstruction algorithm comparing the results of bone-to-implant contact (BIC) evaluated by means of µCT with histomorphometry data. Custom-made conical titanium alloyed (Ti6Al4V) implants were inserted in the distal tibial bone of female Sprague-Dawley rats. Different surface configurations were examined: Ti6Al4V implants with plasma-polymerized allylamine (PPAAm) coating and plasma-polymerized ethylenediamine (PPEDA) coating as well as implants without surface coating. After six weeks postoperatively, tibiae were explanted and BIC was determined by µCT (3D) and afterwards by histomorphometry (2D). In comparison to uncoated Ti6Al4V implants demonstrating low BIC of 32.4% (histomorphometry) and 51.3% (µCT), PPAAm and PPEDA coated implants showed a nonsignificant increase in BIC (histomorphometry: 45.7% and 53.5% and µCT: 51.8% and 62.0%, resp.). Mean BIC calculated by µCT was higher for all surface configurations compared to BIC detected by histomorphometry. Overall, a high correlation coefficient of 0.70 () was found between 3D and 2D quantification of BIC. The μCT analysis seems to be suitable as a nondestructive and accurate 3D imaging method for the evaluation of the bone-implant interface.
- ItemSurface-assisted laser desorption/ionization mass spectrometry using ordered silicon nanopillar arrays(Cambridge : Royal Society of Chemistry, 2014) Alhmoud, Hashim Z.; Guinan, Taryn M.; Elnathan, Roey; Kobus, Hilton; Voelcker, Nicolas H.Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is ideally suited for the high-throughput analysis of small molecules in bodily fluids (e.g. saliva, urine, and blood plasma). A key application for this technique is the testing of drug consumption in the context of workplace, roadside, athlete sports and anti-addictive drug compliance. Here, we show that vertically-aligned ordered silicon nanopillar (SiNP) arrays fabricated using nanosphere lithography followed by metal-assisted chemical etching (MACE) are suitable substrates for the SALDI-MS detection of methadone and small peptides. Porosity, length and diameter are fabrication parameters that we have explored here in order to optimize analytical performance. We demonstrate the quantitative analysis of methadone in MilliQ water down to 32 ng mL-1. Finally, the capability of SiNP arrays to facilitate the detection of methadone in clinical samples is also demonstrated.
- ItemProteome-wide analysis reveals an age-associated cellular phenotype of in situ aged human fibroblasts(Orchard Park : Impact Journals, 2014) Waldera-Lupa, Daniel M.; Kalfalah, Faiza; Florea, Ana-Maria; Sass, Steffen; Kruse, Fabian; Rieder, Vera; Tigges, Julia; Fritsche, Ellen; Krutmann, Jean; Busch, Hauke; Boerries, Melanie; Meyer, Helmut E.; Boege, Fritz; Theis, Fabian; Reifenberger, Guido; StĂ¼hle, KaiWe analyzed an ex vivo model of in situ aged human dermal fibroblasts, obtained from 15 adult healthy donors from three different age groups using an unbiased quantitative proteome-wide approach applying label-free mass spectrometry. Thereby, we identified 2409 proteins, including 43 proteins with an age-associated abundance change. Most of the differentially abundant proteins have not been described in the context of fibroblasts' aging before, but the deduced biological processes confirmed known hallmarks of aging and led to a consistent picture of eight biological categories involved in fibroblast aging, namely proteostasis, cell cycle and proliferation, development and differentiation, cell death, cell organization and cytoskeleton, response to stress, cell communication and signal transduction, as well as RNA metabolism and translation. The exhaustive analysis of protein and mRNA data revealed that 77 % of the age-associated proteins were not linked to expression changes of the corresponding transcripts. This is in line with an associated miRNA study and led us to the conclusion that most of the age-associated alterations detected at the proteome level are likely caused post-transcriptionally rather than by differential gene expression. In summary, our findings led to the characterization of novel proteins potentially associated with fibroblast aging and revealed that primary cultures of in situ aged fibroblasts are characterized by moderate age-related proteomic changes comprising the multifactorial process of aging.
- ItemRaf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells(London : Nature Publishing Group, 2012) Sanges, C.; Scheuermann, C.; Zahedi, R.P.; Sickmann, A.; Lamberti, A.; Migliaccio, N.; Baljuls, A.; Marra, M.; Zappavigna, S.; Reinders, J.; Rapp, U.; Abbruzzese, A.; Caraglia, M.; Arcari, P.We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
- ItemProdukthaftung in der Apotheke(Stuttgart : Deutscher Apotheker Verlag, 2005) Diebold, Steffen M.Stellt ein Apotheker Arzneimittel, Medizinprodukte oder andere apothekenĂ¼bliche Waren (z.B. Kosmetika oder Mixturen nach Angaben von Heilpraktikern, Homöopathen oder Anthroposophen) in seiner Offizin selbst her, so haftet er (grundsätzlich) einem dadurch ggf. geschädigten Kunden (auch) nach den Vorschriften des Produkthaftungsgesetzes. Diese Haftung besteht unabhängig vom eigenen Verschulden. Sie greift auch bei der Einfuhr von Arzneimitteln aus Staaten, die nicht zum Europäischen Wirtschaftsraum gehören, sowie bei Import und Abgabe von aus Drittländern eingefĂ¼hrten und im Geltungsbereich des Arzneimittelgesetzes (AMG) nicht zugelassenen Fertigarzneimitteln nach § 73 (3) AMG.
- ItemRegulation of the tumor-suppressor function of the class III phosphatidylinositol 3-kinase complex by ubiquitin and SUMO(Basel : MDPI, 2014) Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.
- ItemOxaliplatin-DNA adduct formation in white blood cells of cancer patients(London : Nature Publishing Group, 2008) Pieck, A.C.; Drescher, A.; Wiesmann, K.G.; Messerschmidt, J.; Weber, G.; Strumberg, D.; Hilger, R.A.; Scheulen, M.E.; Jaehde, U.In this study, we investigated the kinetics of oxaliplatin-DNA adduct formation in white blood cells of cancer patients in relation to efficacy as well as oxaliplatin-associated neurotoxicity. Thirty-seven patients with various solid tumours received 130 mg m−2 oxaliplatin as a 2-h infusion. Oxaliplatin-DNA adduct levels were measured in the first cycle using adsorptive stripping voltammetry. Platinum concentrations were measured in ultrafiltrate and plasma using a validated flameless atomic absorption spectrometry method. DNA adduct levels showed a characteristic time course, but were not correlated to platinum pharmacokinetics and varied considerably among individuals. In patients showing tumour response, adduct levels after 24 and 48 h were significantly higher than in nonresponders. Oxaliplatin-induced neurotoxicity was more pronounced but was not significantly different in patients with high adduct levels. The potential of oxaliplatin-DNA adduct measurements as pharmacodynamic end point should be further investigated in future trials.
- ItemAutophagy-related deubiquitinating enzymes involved in health and disease(Basel : MDPI, 2015) El Magraoui, Fouzi; Reidick, Christina; Meyer, Hemut E.; Platta, Harald W.Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-receptors. Ubiquitination plays a central role in this process, because it regulates early signaling events during the induction of autophagy and is also used as a degradation-tag on the potential autophagic cargo protein. Here, we review how the ubiquitin-dependent steps of autophagy are balanced or counteracted by deubiquitination events. Moreover, we highlight the functional role of the corresponding deubiquitinating enzymes and discuss how they might be involved in the occurrence of cancer, neurodegenerative diseases or infection with pathogenic bacteria.
- ItemUsing SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice(Rockville : American Society for Biochemistry and Molecular Biology, 2015) Ota, Asuka; Kovary, Kyle M.; Wu, Olivia H.; Ahrends, Robert; Shen, Wen-Jun; Costa, Maria J.; Feldman, Brian J.; Kraemer, Fredric B.; Teruel, Mary N.Insulin resistance (IR) underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of IR, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in IR in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly used insulin-resistant mouse model, Lepr(db/db), and from C57BL/6J control mice. The most differentially expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVCs) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVCs to preadipocyte cell culture models made insulin resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNFα. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types.
- ItemA size dependent evaluation of the cytotoxicity and uptake of nanographene oxide(London [u.a.] : RSC, 2015) Mendes, Rafael Gregorio; Koch, Britta; Bachmatiuk, Alicja; Ma, Xing; Sanchez, Samuel; Damm, Christine; Schmidt, Oliver G.; Gemming, Thomas; Eckert, JĂ¼rgen; RĂ¼mmeli, Mark H.Graphene oxide (GO) has attracted great interest due to its extraordinary potential for biomedical application. Although it is clear that the naturally occurring morphology of biological structures is crucial to their precise interactions and correct functioning, the geometrical aspects of nanoparticles are often ignored in the design of nanoparticles for biological applications. A few in vitro and in vivo studies have evaluated the cytotoxicity and biodistribution of GO, however very little is known about the influence of flake size and cytotoxicity. Herein, we aim at presenting an initial cytotoxicity evaluation of different nano-sized GO flakes for two different cell lines (HeLa (Kyoto) and macrophage (J7742)) when they are exposed to samples containing different sized nanographene oxide (NGO) flakes (mean diameter of 89 and 277 nm). The obtained data suggests that the larger NGO flakes reduce cell viability as compared to smaller flakes. In addition, the viability reduction correlates with the time and the concentration of the NGO nanoparticles to which the cells are exposed. Uptake studies were also conducted and the data suggests that both cell lines internalize the GO nanoparticles during the incubation periods studied.