Untersuchung zur Beteiligung von Proteinkinase A und Tyrosinkinasen an der Calcium-abhängigen Signaltransduktion von Glucagon-like Peptide 1(7-36)amid in Insulin-sezernierenden Zellen des endokrinen Pankreas

Glucagon-like Peptide-1(7-37)/(7-36)amid, GLP-1, ist ein insulinotropes intestinales Peptid-Hormon, das vor allem wegen der Verstärkung der Glukose-abhängigen Insulinsekretion, ein großes Potential bei der Therapie des Diabetes mellitus hat. Die Signaltransduktion von GLP-1 ist in weiten Teilen noch nicht geklärt. Bisherige Studien ergaben, dass GLP-1 an den G-Protein-gekoppelten GLP-1-Rezeptor bindet. Über Aktivierung der Adenylatzyklase erhöht sich der cAMP-Spiegel. Letztlich kommt es durch unbekannte Schritte zu einem Anstieg der intrazellulären Calciumkonzentration und zu einer erhöhten Insulinsekretion. Oftmals sind bei der Signaltransduktion Proteinkinasen wie die cAMP-abhängige Proteinkinase A oder Tyrosinkinasen beteiligt, so dass dies auch bei der GLP-1-induzierten Calciumerhöhung und Insulinsekretion vermutet wurde. Bisherige Arbeiten wurden oft ungeeignete ß-Zellmodelle ausgeführt, verwendet zum Beispiel nur ein Proteinkinase A-Hemmstoff und kamen so zu unterschiedlichen Ergebnissen über die Bedeutung von Proteinkinasen bei der Signaltransduktion von GLP-1. Ziel dieser Arbeit war es, die Beteiligung von Proteinkinase A und Tyrosinkinasen an der Calcium-abhängigen Signaltransduktion von Glucagon-like Peptide 1(7-36)amid in Insulin-sezernierenden Zellen des endokrinen Pankreas zu untersuchen. Dazu wurde 1.) eines geeigneten ß-Zellmodell, hier die Glukose-sensible INS-1-Zellen sowie ß-Zellen in präparierten Langerhanssche Inseln der Maus, und 2.) verschiedene, angemessene Proteinkinasehemmstoffe verwendet. Mit den Fluoreszenzfarbstoffen Fura-2 bzw. Bisoxonol wurden die intrazelluläre Calciumkonzentrationen an beiden Zelltypen sowie Veränderungen im Membranpotential der INS-1-Zellen gemessen. Um Artefakte am Rezeptor auszuschließen, wurde neben GLP-1 durch Forskolin die Adenylatzyklase direkt aktiviert. Weder durch den selektiven Proteinkinase A-Hemmstoff KT5720 (bis zu 20µM) noch Rp-cAMPs (bis 1mM) konnte eine Hemmung der GLP-1- oder Forskolin-Wirkung erzielt werden. Der dritte Proteinkinase A-Inhibitor H-89 selbst führte konzentrationsabhängig (bis 40µM) zu einer deutlichen Reduktion der Glukose-induzierten Effekte, wie den Calciumanstieg oder den Calciumoszillationen der Langerhansschen Inseln. Erst durch lange Inkubationszeiten mit deutlich höheren als sonst verwendeten Konzentrationen ließen sich die GLP-1 bzw. Forskolin-induzierte Erhöhung des Calciumspiegels sowie die Membrandepolarisation leicht reduzieren, so dass es sich dabei eher um einen sekundären Effekt infolge des verminderten Glukoseeinflusses zu handeln scheint. Insgesamt zeigte die fehlende Inhibition durch die drei Hemmstoffe, dass die Proteinkinase A an der Signaltransduktion von GLP-1 bzw. Forskolin zur Erhöhung des intrazellulären Calciumspiegels nicht beteiligt ist. Die Tyrosinkinase-Hemmstoffe Genistein (bis 100µM) und Herbimycin A (bis 20µM) führten beide, Genistein mehr als Herbimycin A, zu konzentrationsabhängigen, massiven Veränderungen der Glukose-abhängigen Calciumerhöhung sowie der Calciumoszillationen. Die Ursache hierfür, ob es sich dabei zum Beispiel um eine spezifische Tyrosinkinase-Hemmung oder vielleicht einen unspezifischen toxischen Effekt handelt, werden in der Literatur kontrovers diskutiert. In Relation zu diesen starken Veränderungen der Glukosewirkung ließ sich bei keinem der beiden Hemmstoffe eine isolierte Inhibition des GLP-1 oder Forskolin-Effektes zeigen. Auch der potente, unspezifische Proteinkinasehemmstoff Staurosporin bei relevanter Dosierung zeigte keine Beeinflussung der durch GLP-1 und Forskolin bedingten Calciumerhöhung sowie der Membrandepolarisation. Insgesamt zeigt dies Arbeit, dass für den GLP-1-induzierten Anstieg der intrazellulären Calciumkonzentration und der Membrandepolarisation weder Proteinkinase A noch Tyrosinkinasen, eher sogar keine Proteinkinase eine entscheidende Rolle spielt. An der möglichen Signaltransduktion dieser GLP-1-Effekte könnten ein nicht selektiver Kationenkanal über cAMP alleinig (NSCC) oder in Kombination mit einer Hyperpolarisation (HCN) ebenso wie der cAMP-regulierte Ras Guanin-Nucleotid-Austausch-Faktor, Epac, beteiligt sein. Um den genauen Mechanismus, unter anderem welche Ionenkanäle beeinflusst werden, aufzuschlüsseln, sind weitere Untersuchungen notwendig. Zudem ist zu erwähnen, dass in der Literatur ebenfalls ein Calcium-unabhängiger Anteile der GLP-1-induzierten Insulinsekretion beschrieben ist. So ist eine Beteiligung von Proteinkinasen distal der Calciumerhöhung, zum Beispiel durch Vergrößerung des Reservepools der Insulingranula oder einer Tyrosinkinase-abhängig verstärkter Exozytose, durchaus mit den Ergebnissen dieser Arbeit vereinbar

Deregulierung in Deutschland : eine empirische Analyse.

Deregulierung; Deutschland;

Limited value of 18F-FDG PET/CT and S-100B tumour marker in the detection of liver metastases from uveal melanoma compared to liver metastases from cutaneous melanoma

Purpose: The objective of this study was to evaluate the value of 18F-FDG PET/CT and S-100B tumour marker for the detection of liver metastases from uveal melanoma in comparison to liver metastases from cutaneous melanoma. Methods: A retrospective evaluation was conducted of 27 liver metastases in 13 patients with uveal melanoma (UM) (mean age: 56.8, range: 30-77) and 43 liver metastases in 14 patients (mean age: 57.9, range: 40-82) with cutaneous melanoma (CM) regarding size and FDG uptake by measuring the maximum standardized uptake value (SUVmax). S-100B serum tumour markers were available in 20 patients. Cytology, histology, additional morphological imaging and follow-up served as reference standard. In nine patients liver metastases were further evaluated histologically regarding GLUT-1 and S-100 receptor expression and regarding epithelial or spindle cell growth pattern. Results: Of 27 liver metastases in 6 of 13 patients (46%) with UM, 16 (59%) were FDG negative, whereas all liver metastases from CM were positive. Liver metastases from UM showed significantly (p < 0.001) lower SUVmax (mean: 3.5, range: 1.5-13.4) compared with liver metastases from CM (mean: 6.6, range: 2.3-15.3). In four of six (66.7%) patients with UM and liver metastases S-100B was normal and in two (33.3%) increased. All PET-negative liver metastases were detectable by morphological imaging (CT or MRI). S-100B was abnormal in 13 of 14 patients with liver metastases from CM. S-100B values were significantly higher (p = 0.007) in the CM patient group (mean S-100B: 10.9μg/l, range: 0.1-115μg/l) compared with the UM patients (mean: 0.2μg/l, range: 0.0-0.5μg/l). Histological work-up of the liver metastases showed no obvious difference in GLUT-1 or S-100 expression between UM and CM liver metastases. The minority (36%) of patients with UM had extrahepatic metastases and the majority (86%) of patients with CM had extrahepatic metastases, respectively. There was a close to significant trend to better survival of UM patients compared with CM patients (p = 0.06). Conclusion: FDG PET/CT and serum S-100B are not sensitive enough for the detection of liver metastases from UM, whereas liver metastases from cutaneous melanoma are reliably FDG positive and lead regularly to increased S-100B tumour markers. The reason for the lower FDG uptake in UM liver metastases remains unclear. We recommend to perform combined contrast-enhanced PET/CT in order to detect FDG-negative liver metastases from U

A quantitative explanation of the observed population of Milky Way satellite galaxies

We revisit the well known discrepancy between the observed number of Milky Way (MW) dwarf satellite companions and the predicted population of cold dark matter (CDM) sub-halos, in light of the dozen new low luminosity satellites found in SDSS imaging data and our recent calibration of the SDSS satellite detection efficiency, which implies a total population far larger than these dozen discoveries. We combine a dynamical model for the CDM sub-halo population with simple, physically motivated prescriptions for assigning stellar content to each sub-halo, then apply observational selection effects and compare to the current observational census. As expected, models in which the stellar mass is a constant fraction F(Omega_b/Omega_m) of the sub-halo mass M_sat at the time it becomes a satellite fail for any choice of F. However, previously advocated models that invoke suppression of gas accretion after reionization in halos with circular velocity v_c <~ 35 km/s can reproduce the observed satellite counts for -15 < M_V < 0, with F ~ 10^{-3}. Successful models also require strong suppression of star formation BEFORE reionization in halos with v_c <~ 10 km/s; models without pre-reionization suppression predict far too many satellites with -5 < M_V < 0. Our models also reproduce the observed stellar velocity dispersions ~ 5-10 km/s of the SDSS dwarfs given the observed sizes of their stellar distributions, and model satellites have M(<300 pc) ~ 10^7 M_sun as observed even though their present day total halo masses span more than two orders of magnitude. Our modeling shows that natural physical mechanisms acting within the CDM framework can quantitatively explain the properties of the MW satellite population as it is presently known, thus providing a convincing solution to the `missing satellite' problem.Comment: 18 pages, 14 figures, accepted to ApJ. Minor changes following referees repor

Minimally invasive, imaging guided virtual autopsy compared to conventional autopsy in foetal, newborn and infant cases: study protocol for the paediatric virtual autopsy trial

BACKGROUND: In light of declining autopsy rates around the world, post-mortem MR imaging is a promising alternative to conventional autopsy in the investigation of infant death. A major drawback of this non-invasive autopsy approach is the fact that histopathological and microbiological examination of the tissue is not possible. The objective of this prospective study is to compare the performance of minimally invasive, virtual autopsy, including CT-guided biopsy, with conventional autopsy procedures in a paediatric population. METHODS/DESIGN: Foetuses, newborns and infants that are referred for autopsy at three different institutions associated with the University of Zurich will be eligible for recruitment. All bodies will be examined with a commercial CT and a 3 Tesla MRI scanner, masked to the results of conventional autopsy. After cross-sectional imaging, CT-guided tissue sampling will be performed by a multifunctional robotic system (Virtobot) allowing for automated post-mortem biopsies. Virtual autopsy results will be classified with regards to the likely final diagnosis and major pathological findings and compared to the results of conventional autopsy, which remains the diagnostic gold standard. DISCUSSION: There is an urgent need for the development of alternative post-mortem examination methods, not only as a counselling tool for families and as a quality control measure for clinical diagnosis and treatment but also as an instrument to advance medical knowledge and clinical practice. This interdisciplinary study will determine whether virtual autopsy will narrow the gap in information between non-invasive and traditional autopsy procedures. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01888380

Structure of Complement Component C2a: Implications for Convertase Formation and Substrate Binding

SummaryC2a provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. We determined two crystal structures of full-length C2a, with and without a pseudo ligand bound. Both structures reveal a near-active conformation of the catalytic center of the serine protease domains, while the von Willebrand factor A-type domains display an intermediate activation state of helix α7 with an open, activated metal-ion-dependent adhesion site. The open adhesion site likely serves to enhance the affinity for the ligand C4b, similar to “inside-out” signaling in integrins. Surprisingly, the N-terminal residues of C2a are buried in a crevice near helix α7, indicative of a structural switch between C2 and C2a. Extended loops on the protease domain possibly envelop the protruding anaphylatoxin domain of the substrate C3. Together with a putative substrate-induced completion of the oxyanion hole, this may contribute to the high substrate specificity of the convertases

Inflammasome and toll-like receptor signaling in human monocytes after successful cardiopulmonary resuscitation

BACKGROUND: Whole body ischemia-reperfusion injury (IRI) after cardiopulmonary resuscitation (CPR) induces a generalized inflammatory response which contributes to the development of post-cardiac arrest syndrome (PCAS). Recently, pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and inflammasomes, have been shown to mediate the inflammatory response in IRI. In this study we investigated monocyte PRR signaling and function in PCAS. METHODS: Blood samples were drawn in the first 12 hours, and at 24 and 48 hours following return of spontaneous circulation in 51 survivors after cardiac arrest. Monocyte mRNA levels of TLR2, TLR4, interleukin-1 receptor-associated kinase (IRAK)3, IRAK4, NLR family pyrin domain containing (NLRP)1, NLRP3, AIM2, PYCARD, CASP1, and IL1B were determined by real-time quantitative PCR. Ex vivo cytokine production in response to stimulation with TLR ligands Pam(3)CSK(4) and lipopolysaccharide (LPS) was assessed in both whole blood and monocyte culture assays. Ex vivo cytokine production of peripheral blood mononuclear cells (PBMCs) from a healthy volunteer in response to stimulation with patients’ sera with or without LPS was assessed. The results were compared to 19 hemodynamically stable patients with coronary artery disease. RESULTS: Monocyte TLR2, TLR4, IRAK3, IRAK4, NLRP3, PYCARD and IL1B were initially upregulated in patients following cardiac arrest. The NLRP1 and AIM2 inflammasomes were downregulated in resuscitated patients. There was a significant positive correlation between TLR2, TLR4, IRAK3 and IRAK4 expression and the degree of ischemia as assessed by serum lactate levels and the time until return of spontaneous circulation. Nonsurvivors at 30 days had significantly lower mRNA levels of TLR2, IRAK3, IRAK4, NLRP3 and CASP1 in the late phase following cardiac arrest. We observed reduced proinflammatory cytokine release in response to both TLR2 and TLR4 activation in whole blood and monocyte culture assays in patients after CPR. Sera from resuscitated patients attenuated the inflammatory response in cultured PBMCs after co-stimulation with LPS. CONCLUSIONS: Successful resuscitation from cardiac arrest results in changes in monocyte pattern recognition receptor signaling pathways, which may contribute to the post-cardiac arrest syndrome. TRIAL REGISTRATION: The trial was registered in the German Clinical Trials Register (DRKS00009684) on 27/11/2015. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13054-016-1340-3) contains supplementary material, which is available to authorized users

Guidelines for pre-operative cardiac risk assessment and perioperative cardiac management in non-cardiac surgery : the Task Force for Preoperative Cardiac Risk Assessment and Perioperative Cardiac Management in Non-cardiac Surgery of the European Society of Cardiology (ESC) and endorsed by the European Society of Anaesthesiology (ESA)

Non-cardiac surgery; Pre-operative cardiac risk assessment; Pre-operative cardiac testing; Pre-operative coronary artery revascularization; Perioperative cardiac management; Renal disease; Pulmonary disease; Neurological disease; Anaesthesiology; Post-operative cardiac surveillanc