TY - JOUR A1 - Sanges, C. A1 - Scheuermann, C. A1 - Zahedi, R. P. A1 - Sickmann, A. A1 - Lamberti, A. A1 - Migliaccio, N. A1 - Baljuls, A. A1 - Marra, M. A1 - Zappavigna, S. A1 - Reinders, J. A1 - Rapp, U. A1 - Abbruzzese, A. A1 - Caraglia, M. A1 - Arcari, P. T1 - Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells JF - Cell Death and Disease N2 - 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. KW - EF-1A KW - Raf kinases KW - signal transduction KW - apoptosis KW - ubiquitin KW - mass spectrometry Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124149 VL - 3 IS - e276 ER - TY - JOUR A1 - Dusik, Verena A1 - Senthilan, Pingkalai R. A1 - Mentzel, Benjamin A1 - Hartlieb, Heiko A1 - Wülbeck, Corina A1 - Yoshii, Taishi A1 - Raabe, Thomas A1 - Helfrich-Förster, Charlotte T1 - The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock JF - PLoS Genetics N2 - All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways. KW - in vitro kinase assay KW - biological locomotion KW - circadian oscillators KW - MAPK signaling cascades KW - circadian rhythms KW - drosophila melanogaster KW - neurons KW - phosphorylation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119433 SN - 1553-7404 VL - 10 IS - 8 ER - TY - JOUR A1 - Lückerath, Katharina A1 - Lapa, Constantin A1 - Malzahn, Uwe A1 - Samnick, Samuel A1 - Einsele, Herrmann A1 - Buck, Andreas K. A1 - Herrmann, Ken A1 - Knop, Stefan T1 - 18FDG-PET/CT for prognostic stratification of patients with multiple myeloma relapse after stem cell transplantation N2 - The aim of this study was to investigate the prognostic value of 18F-fluoro-deoxyglucose positron emission tomography–computed tomography (18F-FDG-PET/CT) in 37 patients with a history of multiple myeloma (MM) and suspected or confirmed recurrence after stem cell transplantation (SCT). All patients had been heavily pre-treated. Time to progression (TTP) and overall survival (OS) were correlated to a number of different PET-derived as well as clinical parameters. Impact on patient management was assessed. Absence of FDG-avid MM foci was a positive prognostic factor for both TTP and OS (p<0.01). Presence of >10 focal lesions correlated with both TTP (p<0.01) and OS (p<0.05). Interestingly, presence of >10 lesions in the appendicular skeleton proved to have the strongest association with disease progression. Intensity of glucose uptake and presence of extramedullary disease were associated with shorter TTP (p=0.037 and p=0.049, respectively). Manifestations in soft tissue structures turned out to be a strong negative predictor for both, TTP and OS (p<0.01, respectively). PET resulted in a change of management in 30% of patients. Our data underline the prognostic value of 18F-FDG-PET/CT in MM patients also in the setting of post-SCT relapse. PET/CT has a significant impact on patient management. KW - 18FDG-PET/CT KW - Multiple myeloma KW - molecular imaging KW - FDG-PET/CT Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-113107 ER - TY - JOUR A1 - Klement, Rainer Johannes A1 - Frobel, Thomas A1 - Albers, Torsten A1 - Fikenzer, Sven A1 - Prinzhausen, Jan A1 - Kämmerer, Ulrike T1 - A pilot case study on the impact of a self-prescribed ketogenic diet on biochemical parameters and running performance in healthy and physically active individuals N2 - Background: Ketogenic diets (KDs) have gained some popularity not only as effective weight-loss diets and treatment options for several diseases, but also among healthy and physically active individuals for various reasons. However, data on the effects of ketosis in the latter group of individuals are scarce. We therefore collected pilot data on the physiological response to a self-prescribed ketogenic diet lasting 5-7 weeks in a small cohort of healthy and physically active individuals. Methods: Twelve subjects (7 males, 5 females, age 24-60 years) who followed moderate to intensive exercise routines underwent blood testing, bioelectrical impedance analysis (BIA) and spiroergometry during an incremental treadmill test. On the next day, they went on a self-prescribed KD for a median of 38 days (range 35-50 days), after which the same tests were performed again. Ketosis was self-monitored by urinary ketone strips. Subjective feeling during the diet was assessed by a questionnaire after the intervention. Due to the small and heterogenous sample, the results are interpreted in the context of the already existing literature. Results: The KDs were tolerated well by the majority of individuals. Impaired recovery from exercise remained the most frequently reported side effect until the end of the study. Most blood parameters remained stable during the intervention. However, there were significant elevations of total and LDL cholesterol concentrations (p<0.01) and a trend towards increased HDL-cholesterol (p=0.05). The drastic reduction of carbohydrates had no statistically significant influence on running performance judged by the time to exhaustion, VO2max and respiratory compensation points. BIA measurements showed significant increases in phase angle (p=0.01) indicating improvements of body composition with an estimated decrease of 3.4 kg of fat mass (p=0.002) and gain of 1.3 kg of fat free mass. We discuss the validity of these estimates taking into account a possibly altered hydration status due to the KD. Conclusions: Active healthy individuals will probably experience no major problems during a short term KD lasting several weeks. The drastically reduced carbohydrate content of the diet seems to be no limiting factor for running performance. In addition, improvements in body composition can be expected. While most biochemical parameters are not influenced by the diet, there seems to be an impact on the blood lipid profile that could be considered problematic with respect to cardiovascular disease risk. However, the predictive role of cholesterol levels alone in individuals undergoing regular physical activity remains to be elucidated. KW - Fettgehalt KW - Blut KW - Ketogene Kost KW - High fat diet KW - exercise KW - bioimpedance analysis KW - blood lipids; fat loss; running performance Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78901 ER - TY - JOUR A1 - Puetz, Stephanie M. A1 - Vogiatzi, Fotini A1 - Stiewe, Thorsten A1 - Sickmann, Albert T1 - Malignant transformation in a defined genetic background: proteome changes displayed by 2D-PAGE N2 - Background: Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER). Results: The stepwise malignant transformation of human fibroblasts was analyzed on the protein level by differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins. The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen (PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90 were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2 (PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we confirmed our 2D-PAGE results by Western Blot. Conclusion: These findings give further hints for intriguing interactions between the p16-RB pathway, the mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of tumorigenesis. KW - Medizin Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68321 ER -