Axiales Patterning und seine Steuerung in der Embryogenese eines basalen Metazoen, Hydractinia echinata

In dieser Arbeit konnten erstmals Transgene von Hydractinia echinata unter der Kontrolle der nativen regulatorischen Regionen von unterschiedlichen Genen erzeugt und Expressions-studien mithilfe des Reportergen eGFP durchgeführt werden. Es wurden zwei Arten von Konstrukten erzeugt: a) solche unter möglichst ubiquitär aktiven Promotoren und b) solche, die den Promotor eines funktionell zu untersuchenden Gens erhielten. Im Genom von Hydractinia existieren multiple Gene für Aktin, die trotz der Identität der Proteinstruktur (> 99%) unterschiedliche 5`- und 3`regulatorische Regionen aufweisen. Die Aktivität der regulatorischen Regionen der drei zytoplasmatischen Beta-Aktingene HeActI, HeActII und HeActIII konnten durch erfolgreiche Expression von eGFP gezeigt werden. Die Expressionsmuster belegen, dass diese drei ß-Aktine unterschiedlich in Hydractinia exprimiert werden. Ausschließlich HeActII zeigte eine ubiquitäre Expression. Als ein weiteres ubiquitär exprimiertes Gen wurde HeEF1alpha verwendet. Die vorliegende Studie hat gezeigt, dass in Hydractinia Kontrollgene konserviert sind, die in den Hauptsignalwegen der höheren Metazoen Entwicklung kontrollieren. Deren mögliche Funktion in diesem basalen Metazoen wurde mithilfe von Funktionsanalysen durch den Einsatz transgener Techniken nachgegangen. Damit gelang im Rahmen dieser Arbeit ein Einstieg in die Funktionsanalyse von Proteinen in Hydractinia. Dafür wurden die regulatorischen Regionen des ubiquitär exprimierten Gens HeActII zur Entwicklung eines Expressionskonstruktes ausgewählt, um die mögliche Funktion eines Proteins durch ektopische Expression in transgenen Hydractinia zu untersuchen. Manche der standardmäßig im Labor eingesetzten transgenen Techniken zur Funktionsanalsye konnten in Hydractinia nicht verwendet werden. Zwei experimentelle Ansätze mittels Überexpression der analysierten Gene Heß-Cat bzw. HeGsc führten zu Gestaltbildungseffekten während der larvalen Entwicklung und in post-metamorphen Stadien von Hydractinia. Mithilfe transgener Technik gelang es erstmals, das Nervensystem von Hydractinia echinata in vivo darzustellen. Es wurden unterschiedliche Nervenzelltypen durch eGFP-Markierung detektierbar. Ein Teil dieser Zellen war bereits aus ICC-Studien bekannt. Hinzu kamen Nervenzellen, die zuvor noch nie beobachtet wurden. Darunter befanden sich sog. giant bipolar neurons mit einer Länge von mindestens 250μm, die im Stolon und auch in der Planulalarve nachgewiesen wurden. Die Ergebnisse weisen auf eine weit grössere Komplexität des Nervensystems dieses einfach gebauten Metazoen hin, als bisher bekannt. Des Weiteren zeigte sich, dass das Nervensystem nicht nur longitudinal, sondern auch transversal zur anterior-posterioren Körperachse der Larve vernetzt ist. Diese neuen Einblicke in das Nervensystem wurden durch Expressionsstudien unter der Kontrolle der genregulatorischen Regionen der beiden ubiquitär exprimierten housekeeping genes HeActII und HeEF1α, sowie durch die des neuralspezifischen Gens HeELAV möglich. Die Expressionsstudien zeigten, dass sich HeELAV ein hochgeeignetes neuralspezifischers Markergen ist, um in vivo die Entwicklung des Nervensystems von Hydractinia zu untersuchen

A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP-modulated NFAT signaling

Alternative splicing is a potent modifier of protein function. Stro mal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcrip tion factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astro cytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and desta bilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteo mics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activ ity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regu late the NFAT signalosome and cAMP-SOCE crosstalk

Delineating endogenous Cushing's syndrome by GC-MS urinary steroid metabotyping

BACKGROUND Diagnosing Cushing's syndrome (CS) is highly complex. As the diagnostic potential of urinary steroid metabolome analysis by gas chromatography-mass spectrometry (GC-MS) in combination with systems biology has not yet been fully exploited, we studied a large cohort of patients with CS. METHODS We quantified daily urinary excretion rates of 36 steroid hormone metabolites. Applying cluster analysis, we investigated a control group and 168 patients: 44 with Cushing's disease (CD) (70% female), 18 with unilateral cortisol-producing adrenal adenoma (83% female), 13 with primary bilateral macronodular adrenal hyperplasia (PBMAH) (77% female), and 93 ruled-out CS (73% female). FINDINGS Cluster-Analysis delineated five urinary steroid metabotypes in CS. Metabotypes 1, 2 and 3 revealing average levels of cortisol and adrenal androgen metabolites included patients with exclusion of CS or and healthy controls. Metabotype 4 reflecting moderately elevated cortisol metabolites but decreased DHEA metabolites characterized the patients with unilateral adrenal CS and PBMAH. Metabotype 5 showing strong increases both in cortisol and DHEA metabolites, as well as overloaded enzymes of cortisol inactivation, was characteristic of CD patients. 11-oxygenated androgens were elevated in all patients with CS. The biomarkers THS, F, THF/THE, and (An + Et)/(11β-OH-An + 11β-OH-Et) correctly classified 97% of patients with CS and 95% of those without CS. An inverse relationship between 11-deoxygenated and 11-oxygenated androgens was typical for the ACTH independent (adrenal) forms of CS with an accuracy of 95%. INTERPRETATION GC-MS based urinary steroid metabotyping allows excellent identification of patients with endogenous CS and differentiation of its subtypes. FUNDING The study was funded by the Else Kröner-Fresenius-Stiftung and the Eva-Luise-und-Horst-Köhler-Stiftung

Persisting Muscle Dysfunction in Cushing's Syndrome Despite Biochemical Remission

CONTEXT Glucocorticoid-induced myopathy is a characteristic symptom of endogenous Cushing's syndrome (CS). Its long-term outcome is largely unknown. OBJECTIVE To evaluate long-term muscle function following the remission of endogenous CS. STUDY DESIGN Observational longitudinal cohort study. SETTING Tertiary care hospitals and a specialized outpatient clinic. PATIENTS As part of the prospective multicenter German Cushing's Registry, we assessed muscle strength in patients with overt endogenous CS. We studied the patients at the time of diagnosis (n = 88), after 6 months (n = 69), and thereafter annually, following surgical remission over a period of up to 4 years (1 year: n = 55; 2 years: n = 34; 3 years: n = 29; 4 years: n = 22). Muscle function was evaluated by hand grip strength and by chair rising test. RESULTS Grip strength was decreased to 83% of normal controls (100%) at the time of diagnosis. It further decreased to 71% after 6 months in remission (P ≤ 0.001) and showed no improvement during further follow-up compared with baseline. Chair rising test performance improved initially (8 seconds at baseline vs 7 seconds after 6 months, P = 0.004) but remained at this reduced level thereafter (7 seconds after 3 years vs 5 seconds in controls, P = 0.038). In multivariate analysis, we identified, as predictors for long-term muscle dysfunction, age, waist-to-hip ratio, and hemoglobin A1c at baseline. Furthermore, muscle strength during follow-up was strongly correlated with quality of life. CONCLUSION This study shows that CS-associated myopathy does not spontaneously resolve during remission. This calls for action to identify effective interventions to improve muscle dysfunction in this setting

Migration and differentiation potential of stem cells in the cnidarian hydractinia analysed in egfp-transgenic animals and chimeras

AbstractTo analyse cell migration and the differentiation potential of migratory stem cells in Hydractinia, we generated animals with an eGFP reporter gene stably expressed and transmitted via the germline. The transgene was placed under the control of two different actin promoters and the promoter of elongation factor-1α. One actin promoter (Act-II) and the EF-1α promoter enabled expression of the transgene in all cells, the other actin promoter (Act-I) in epithelial and gametogenic cells, but not in the pluripotent migratory stem cells. We produced chimeric animals consisting of histocompatible wild type and transgenic parts. When the transgene was under the control of the epithelial cell specific actin-I promoter, non-fluorescent transgenic stem cells immigrated into wild type tissue, stopped migration and differentiated into epithelial cells which then commenced eGFP-expression. Migratory stem cells are therefore pluripotent and can give rise not only to germ cells, nematocytes and nerve cells, but also to epithelial cells. While in somatic cells expression of the act-I promoter was restricted to epithelial cells it became also active in gametogenesis. The act-I gene is expressed in spermatogonia, oogonia and oocytes. In males the expression pattern showed that migratory stem cells are the precursors of both the spermatogonia and their somatic envelopes. Comparative expression studies using the promoters of the actin-II gene and the elongation factor-1α gene revealed the potential of transgenic techniques to trace the development of the nervous system

Numerical study of time-dependent hygrothermal conditions in depressurized crawl space

A finite element based hygrothermal model consisting of several interconnected components with varying number of spatial dimensions was applied to analyze the time-dependent temperature and humidity conditions of a mechanically depressurized and ventilated crawl space. Purpose of the depressurization is to prevent the intrusion of radon or other insanitary particles into indoor air. However, in typical foundation structures the depressurization will cause airflow from soil into the crawl space air and it may convey excessive moisture making the hygrothermal conditions potential for mould growth or other moisture-induced biological damage, which is not considered to be acceptable even with the depressurization. Although in general the forced convection of humidity from soil presumably increases relative humidity in crawl space, significant heat capacity of the ground may warm the air flowing into the crawl space and thus decrease the relative humidity. Overall effect of the depressurization on the conditions in crawl space is therefore not trivial. Because a full-scale three-dimensional finite element analysis of heat, mass and momentum transfer in crawl space and its surroundings would require excessive computational resources, several simplifications were necessary to apply in the model. According to the numerical results, the airflow through drainage layer into crawl space does not seem to have severe effect on the crawl space conditions. Conversely, in cold periods the relative humidity in crawl space is very low because of the air temperature is increased while flowing through the drainage layer.Peer reviewe