Molecular determinants for the palmitoylation of viral and cellular membrane proteins

Deckblatt-Impressum persönlicher Dank Inhaltsverzeichnis Verzeichnis der Abbildungen Abkürzungsverzeichnis Einleitung und Fragestellung Schrifttum Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Veröffentlichungen Danksagung SelbständigkeitserklärungUnter dem Begriff der Palmitoylierung wird eine posttranslationale Modifikation von Proteinen verstanden, wobei in den meisten Fällen ein Palmitinsäurerest über eine Thioesterbindung an einen Cysteinrest des Proteins gebunden wird. Diese Modifikation ist in nahezu allen eukaryotischen Lebensformen weit verbreitet. Während laufend weitere Proteine identifiziert werden können, die in dieser Weise modifiziert sind, bleiben noch immer viele Fragen hinsichtlich der zugrunde liegenden molekularen Mechanismen offen. In den letzten fünf Jahren konnten große Fortschritte bei der Entschlüsselung der zugrunde liegenden Enzyme und dynamischen Regelmechanismen erzielt werden. Spezifische Sequenzmuster für eine Palmitoylierung konnten bislang allerdings nur für cytoplasmatisch vorliegende Proteine entdeckt werden. In integralen Membranproteinen liegt die Palmitoylierungsstelle im Allgemeinen nahe der Grenze zwischen cytoplasmatischer Domäne und Transmembranregion. Ein Vergleich der Aminosäuresequenzen palmitoylierter Proteine zeigt aber keine deutliche Konsensus-Sequenz, was vermuten lässt, dass acylierte Proteine in der cytoplasmatischen Domäne und der Transmembranregion komplexere Informationen für die Palmitoylierung beinhalten. Im Rahmen dieser Arbeit sollte der Einfluss der Transmembranregion und speziell dort vorliegender Aminosäuren auf die Palmitoylierung untersucht werden. Hierzu wurden Chimären aus palmitoylierten und nicht palmitoylierbaren Proteinen hergestellt. Diese Versuche zeigten, dass sowohl der Ersatz von Transmembranregion und cytoplasmatischer Domäne zusammen als auch nur der Transmembranregion des nicht palmitoylierbaren Fusionsproteins des Sendaivirus allein gegen die entsprechenden Domänen der palmitoylierten Proteine CD4, CD8 oder Influenza A-Virus Hämagglutinin eine befriedigende Palmitoylierung der Chimären ermöglichte. Die Transmembranregion muss demnach molekulare Informationen für die Palmitoylierung enthalten. Auch nicht-hydrophobe Aminosäure-Muster scheinen eine Palmitoylierung zu unterstützen. Ein Sequenzvergleich bekannter palmitoylierter Transmembranproteine zeigt ein häufiges Vorliegen der Aminosäure Glycin im cytoplasma-nahen Bereich der Transmembranregion. Austausch der membrannahen Glycin- und Phenylalaninreste in der Transmembranregion führte zu gravierendem Abfall des Palmitoylierungsniveaus, was sich durch die veränderte Ausrichtung des Proteins in der Membran und so gestörte Interaktion mit der membranständigen Palmitoyltransferase erklären lässt. Weiter entfernt liegende Glycinreste hatten keinen Einfluss auf die Acylierung. Nicht-hydrophobe Cytoplasma-nahe Motive in der ansonsten strikt hydrophoben Transmembrandomäne stellen demnach molekulare Signale für eine Palmitoylierung dar.The covalent attachment of fatty acids - commonly palmitic acid - in a thioester-type linkage is a widespread modification of viral and cellular polypeptides. While constantly further palmitoylated proteins are being identified, many questions remain open regarding the molecular mechanisms for palmitoylation. So far specific sequence motives for palmitoylation could be discovered only for cytoplasmic proteins. With integral membrane proteins the hydrocarbon chain is usually bound to cysteine residues located close to the boundary between the transmembrane region and the cytoplasmic tail. Inspection of the amino acids in the vicinity of the acylated cysteine residues reveals no obvious consensus-signal for palmitoylation. This implies that acylated membrane proteins contain complex conformational signals for palmitoylation that are mainly located within the cytoplasmic half of the transmembrane domain, but also involve the cytoplasmic region. Therefore the aim of this study was to resolve the influence of the transmembrane region for palmitoylation as well as the particular contribution of amino acid residues within the transmembrane domain using a series of new chimeric and mutant proteins derived from the acylated proteins Influenza virus haemagglutinin, CD4 and CD8 receptor protein as well as from the non-acylated Sendai virus fusion protein. While Sendai virus fusion protein is not palmitoylated even after introducing of a cysteine residue into the potential acylation region, substitution of the transmembrane domain or both transmembrane and cytoplasmic region for the corresponding regions of Influenza virus haemagglutinin or CD4- or CD8 receptor protein is sufficient for palmitoylation. Non-hydrophobic amino acid residues on the hydrophilic face of the TMD-helix may support palmitoylation of membrane proteins. Sequence alignment of some palmitoylated proteins shows frequent occurrence of glycine in the transmembrane region close to the cytoplasmic tail. Replacement of cytoplasma-near glycine and phenylalanine residues in the transmembrane region leads to a lower acylation rate of the chimerae, while distant glycine residues have no significant influence on palmitoylation rate. This may be due to changes in the protein adjustment in the membrane and therefore disturbed interaction with the membrane-based enzyme palmitoyltransferase. Non-hydrophobic motives in the otherwise strictly hydrophobic transmembrane helix therefore represent molecular signals for palmitoylation

Diagnostic orientation values for ACTH and other parameters for clinically healthy donkeys and mules (insulin, triglycerides, glucose, fructosamines, and ɣ‐GT)

Pituitary pars intermedia dysfunction is the most prevalent endocrine disease in horses. Although donkeys and mules may also be affected, only a few data have been published. Reference values for diagnostic parameters, such as adrenocorticotropic hormone (ACTH), are especially scarce or even lacking. Therefore, in the present study, available data from the literature have been verified and completed to facilitate a reliable diagnosis. Clinical inspections and haematological and biochemical examinations were carried out four times in a three‐month interval (February to November) in 44 donkeys and 31 mules. Data from clinically healthy animals were used as an orientation. Plasma ACTH concentrations showed seasonal changes in both animal groups. However, it was generally higher in donkeys than mules. Although blood glucose (EDTA plasma) showed no difference between groups, serum insulin concentrations were consistently higher in donkeys. Serum fructosamine levels were slightly higher in mules, whereas, in some cases, serum triglyceride levels were considerably higher in donkeys. Serum gamma‐glutamyltransferase showed a striking peak in mules in August, whereas the remaining gamma‐glutamyltransferase values were lower compared to donkeys. By comparing donkeys and mules, the present work reveals differences in various blood parameters which should be considered for diagnoses and future studies

Pre-Ride Biomarkers and Endurance Horse Welfare: Analyzing the Impact of the Elimination of Superoxide Dismutase, δ-Aminolevulinic-Dehydratase, Thiobarbituric Acid Reactive Substances, Iron, and Serum Amyloid A Levels in Elite 160 km Endurance Rides

High elimination rates and concerns for horse welfare are important issues in endurance riding. Improved understanding of the causes of elimination could increase completion rates in this sport. We have identified pre-ride laboratory risk factors that enable an assessment of potential elimination before the ride. A longitudinal cohort study was performed among 49 healthy horses competing in the 160 km endurance ride at the 2016 World Championship of Endurance Riding in Samorin/Slovakia. Blood samples were taken before the event. For statistical evaluation, horses were categorized into three groups: finishers, lame horses, and metabolically eliminated horses. Risk factors were calculated for each group using multinominal logistic regression. δ-Aminolevulinic-dehydratase (ALAD), thiobarbituric acid reactive substances (TBARSs), iron, and serum amyloid A (SAA) were measured and did not show an impact on the race outcome, but elevated pre-ride superoxide dismutase (SOD) was shown to have an effect on lameness elimination (p = 0.011). It might serve as an indicator for withdrawing horses at risk of later elimination before endurance rides, ultimately resulting in lower elimination rates and an increase in overall horse welfare

Wohin des Weges? Regionalentwicklung in Großschutzgebieten

Im Zuge der Einrichtung von Großschutzgebieten in Deutschland wurden mit Nationalparken, Biosphärenreservaten und Naturparken Schutzgebietskategorien etabliert, die vom Grundsatz her unterschiedliche Hauptzielsetzungen aufweisen. In den letzten Jahren scheint es nun allerdings zu einer Überlappung von Aufgabenschwerpunkten zu kommen - insbesondere vor dem Hintergrund der vielfältig zu begreifenden Aufgabe der Regionalentwicklung. Mit einem Schwerpunkt auf den Ländern Hessen, Rheinland-Pfalz und Saarland rücken aktuelle Fragestellungen im Schnittfeld von Großschutzgebieten und Regionalentwicklung in den Mittelpunkt der Betrachtung. Innerhalb des Einführungsbeitrags wird zunächst ein kurzer Überblick über die drei Großschutzgebietskategorien Naturparke, Nationalparke und Biosphärenreservate sowie das mögliche Aufgabenverständnis von Regionalentwicklung gegeben. Anschließend werden zentrale Ergebnisse der weiteren Artikel dargestellt und zusammenfassend eingeordnet - entsprechend dem Zugang: Wohin des Weges? Regionalentwicklung in Großschutzgebieten.When large-scale protected areas were established in Germany, three categories of protected areas were set up: national parks, biosphere reserves and nature parks, each of which in principle pursue different primary objectives. In recent years, however, some overlapping of focuses seems to have emerged - particularly against the background of regional development, which can be understood in a variety of ways. Focusing on the states Hesse, Rhineland-Palatinate and Saarland, this investigation addresses topical issues at the interface between large-scale protected areas and regional development - thus: Where are we heading? Regional development in largescale protected areas

Gegen den Wind - Konfliktlinien beim Ausbau erneuerbarer Energien in Großschutzgebieten am Beispiel der Windenergie in den Naturparken Soonwald-Nahe und Rhein-Westerwald

Auch wenn die Energiewende in weiten Teilen der Bevölkerung große Zustimmung findet, rufen ihre physischen Manifestationen wie Photovoltaik- oder Windkraftanlagen immer wieder lokale Proteste hervor. Großschutzgebiete wie Biosphärenreservate und Naturparke stehen hier vor einer besonderen Herausforderung: Es besteht kein umfänglicher Konsens, ob erneuerbare Energien nun dem Naturschutz dienen oder eher schaden beziehungsweise inwieweit sie einen wichtigen Bestandteil nachhaltiger Regionalentwicklung darstellen. Im Beitrag werden zwei rheinland-pfälzische Naturparke mit und ohne Windkraftnutzung miteinander verglichen. Aus diskurstheoretischer Perspektive, basierend auf einer Zeitungs- und Websiteanalyse, wird beleuchtet, welche Muster in Bezug auf Windkraft und die Argumentation über den Status als Großschutzgebiet zentral verankert sind. Windkraftausbau innerhalb des Naturparks Soonwald-Nahe wird zwar teilweise als negativ bewertet, der Großschutzgebietsstatus jedoch nicht explizit als Ausschlussgrund angesehen. Der Naturpark wird vielmehr als schützens- und erhaltenswerte Referenzgröße gerahmt. Im Fallkontext Rhein-Westerwald erfolgt die Argumentation über den Großschutzgebietsstatus umfangreicher: Hier wird der Naturpark zu einer regionalen, emotionalen Bezugsgröße, dessen "Verschandelung" von den Gegnerinnen und Gegnern als moralisch verwerflich gedeutet wird. In beiden Fällen schließen sich in der Argumentation tendenziell Windkraft und Naturparke aus. Windenergie wird nicht als Potenzial der Regionalentwicklung assoziiert.Although the 'energy transition' is supported by a large proportion of the population, its physical manifestations in the form of photovoltaic installations or wind turbines repeatedly trigger local protests. Large-scale protected areas like biosphere reserves and nature parks face particular challenges in this context. There is no complete consensus about whether renewable energies benefit or harm nature conservation, or about the extent to which they represent an important element of 'sustainable regional development'. This paper compares two nature parks in Rheinland-Pfalz, one with wind turbines and one without. From the perspective of discourse theory, an analysis of newspapers and websites sheds light on centrally anchored discourse patterns related to wind power and arguments about the status of large-scale protected areas. The development of wind power within the Soonwald-Nahe Nature Park is at times judged negatively but its status as a large-scale protected area is not explicitly seen to be grounds for exclusion. The nature park is rather framed as a unit of reference which is worthy of protection and conservation. In the context of the case study of Rhein-Westerwald argumentation about the status of large-scale protected areas is more extensive. The nature park is presented as a regional, emotional reference and its ‘disfigurement’ is construed by opponents as morally reprehensible. In both cases the lines of argument tend to rule out the combination of wind power and nature parks. Wind energy is not associated with regional development potential

Embodied Agents of Life- and Cyberscience : Bericht über ein Symposium der TU Braunschweig und der Universität Bremen

Das internationale Symposium “Embodied Agents of Life- and Cyberscience. Turbulente Körper und soziale Maschinen“ (5. - 7.7.2002) brachte knapp dreißig ForscherInnen aus den Geistes- und Naturwissenschaften zusammen, um Fragen des 'technological embodiment' zu diskutieren. Die konsequent transdisziplinäre Herangehensweise und die sozio-materiale Auseinandersetzung mit aktuellen Technologien ermöglichte ein tiefergehendes Verständnis gegenwärtiger Körperformierungen, Maschinenkonzepte und Technofakte jenseits eindimensionaler Technikeuphorie oder -pessimismus. Den Auftakt der Arbeitstagung bildeten zwei Vorträge international renommierter Wissenschaftsforscherinnen. N. Katherine Hayles (USA) untersuchte Umschreibungen des 'Humanen' in den neuesten Entwicklungen der Robotik-Forschung, während Lucy Suchman (GB) in ‚Embodied Agencies at the Interface’ zeigte, dass die Konzepte von Software-Agenten, Wearable Computing und Intelligenten Häusern klassenhierarchische und geschlechtsspezifische Phantasien reproduzieren. Anschließend wurde in den Arbeitsgruppen „Sozialität mit Menschen und Maschinen”, “Im/Materialität – oder der Mythos vom Verschwinden des Körpers“ und „Emergenz – Formalisierung des Unverfügbaren?“ auf der Grundlage von Positionspapieren diskutiert. Dabei ließen sich zentrale Erkenntnisse zur technologischen Verkörperung und neue Forschungsfragen entwickeln. Insgesamt wurde deutlich, dass eine kritische Gesellschaftsanalyse und Genderforschung wie auch die Technologieentwicklung unabdingbar der Wissenschafts- und Technikforschung bedarf. Gesellschaftstheorie, Körpertheorie und die Techniktheorie bilden ein ‚seamless web’, das nur an der Schnittstelle von Natur- und Geisteswissenschaften adäquat analysiert werden kann. Das Symposium mit seiner innovativen Gestaltung hat erste Schritte in dieser Richtung unternommen und damit den notwendigen Anschluss an die internationale Debatte geleistet.The international symposium “Embodied Agents of Life- and Cyberscience. Turbulente Bodies and Social Machines“ (5. - 7.7.2002) brought together almost 30 researchers from the natural sciences and humanities in order to discuss topics of technological embodiment in the age of technoscience. The consistent transdisciplinary approach and the socio-material engagement in current technologies gave the participants a deeper understanding of present concepts and formations of bodies, machines and artefacts beyond one-dimensional technological enthusiasm and affirmation. The conference started with two lectures from internationally renowned researchers in science and technology studies. In “Computing the Human” N. Katherine Hayles (USA) focussed on reinterpretations of the ‘human’ which occurred in the latest developments in robotic research, while Lucy Suchman (GB) elaborated on the fantasies of class and gender within the concepts of software agents, Wearable Computing and Intelligent Environments in her talk “Embodied Agents at the Interface”. Later, three open discussion groups concentrated on the topics “Sociality between humans and machines”, “Im/Materiality or the myth of dissolution” and “Emergence – formalizing the unavailable”, prepared by position papers of all participants. These debates gave profound insights into the field of technological embodiment and raised new research questions. In summary, the symposium emphasized that critical analyses of society, gender studies and technology design need science and technology studies. Theories of society, the body and technology form a seamless web which can only be explored on the interface of science and humanities. The symposium with its innovative form took first steps in this direction and linked the research in the field of technological embodiment and its gendering to international debates

Age and Hydration of Competing Horses Influence the Outcome of Elite 160 km Endurance Rides

High elimination rates and the concern for horse welfare are important issues in endurance riding. An improved understanding of the causes of elimination could increase completion rates in this sport. We have identified pre-ride risk factors that allow an assessment of potential elimination before the ride. A longitudinal cohort study was performed among 49 healthy horses competing in the 160 km endurance ride at the 2016 World Championship of Endurance Riding in Samorin/Slovakia. Blood samples were drawn before the ride. For statistical evaluation, horses were categorized in three groups: finishers, lame and metabolically eliminated horses. Risk factors were calculated for each group using multinomial logistic regression. A 1% increase in hematocrit levels was associated with a higher OR for elimination (lameness: OR 1.26, p = 0.017; metabolic: OR 1.34, p = 0.010). Furthermore, increased potassium values correlated negatively with the race outcome. For a 1 mmol/l increase in potassium, the lameness OR was 4.21, p = 0.039 and metabolic OR was 1.15, p = 0.848. Eight-year-old horses had a 100% elimination rate and survival analyses showed a significantly higher hazard for elimination (p = 0.025). We thus conclude that age and hydration affect the outcome of elite endurance rides. Further investigation of age as a risk factor seems to be clinically relevant and adjustments of FEI qualification modes may be appropriate

Evaluation of the diagnostic accuracy of a point-of-care device to measure concentrations of nonesterified fatty acids in serum and whole blood

The objective of this study was to compare measurements of nonesterified fatty acids (NEFA) between the gold standard diagnostic laboratory method and a handheld NEFA meter (Qucare Pro meter, DFI Co. Ltd.). Three experiments were conducted to study the usability of the meter. In experiment 1 we compared results of the meter obtained from measurements in serum and whole blood with results of the gold standard method. Based on the results of experiment 1 we compared the results measured by the meter in whole blood with results obtained from the gold standard method on a larger scale, as we wanted to omit the step of centrifugation with the cow-side test. In experiment 3 we determined the influence of ambient temperature on measurements. Overall, blood samples of 231 cows were collected between 14 and 20 d in milk. The Spearman correlation coefficients (ρ) were calculated and Bland-Altman plots were created to compare the accuracy of the NEFA meter with the gold standard. In addition, in experiment 2 receiver operating characteristic (ROC) curve analyses were performed, to define thresholds for the NEFA meter to detect cows with a NEFA concentration above 0.3, 0.4, and 0.7 mEq/L. In experiment 1, there was a high correlation between NEFA concentrations in whole blood and serum determined by the NEFA meter and the gold standard (ρ = 0.90 for measurement in whole blood; ρ = 0.93 for measurement in serum). In experiment 2, the measurement in whole blood with the NEFA meter was compared with the gold standard. Despite a lower correlation (ρ = 0.79) the ROC curve analyses revealed a high specificity and a moderate sensitivity for lower cut-points (i.e., 0.3 and 0.4 mEq/L). The NEFA meter underestimated especially high concentration of >0.7 mEq/L. Considering thresholds of 0.3, 0.4, and 0.7 mEq/L measured by the gold standard test, sensitivity and specificity were 59.1% and 96.7%, 79.0% and 95.4%, and 86.4% and 95.6%, respectively, when using 0.3, 0.3, and 0.4 mEq/L as thresholds for the NEFA meter. Accuracy was 74.1%, 88.3%, and 93.8% for the 3 thresholds tested. Experiment 3 showed that measurements should be conducted at approximately 21°C (ρ = 0.73) as correlations were poor at 6.2°C and 15.1°C (ρ = 0.18 and 0.22, respectively)

Metabolic changes associated with two endocrine abnormalities in dogs : elevated fructosamine and low thyroxine

Introduction Metabolomics studies in canine endocrine abnormalities are sparse and basic information on these abnormalities must be generated. Objectives To characterize the metabolic changes associated with elevated fructosamine, reflecting poor glycemic control, and low thyroxine, a thyroid hormone controlling metabolism. Methods Leftovers of clinical serum samples; 25 controls, 79 high fructosamine, and 47 low thyroxine, were analyzed using H-1 NMR and differences were evaluated using Firth logistic regression. Results Both high fructosamine and low thyroxine were associated with changes in concentrations of multiple metabolites, including glycoprotein acetyls and lipids. Conclusion These findings suggest promising makers for further research and clinical validation.Peer reviewe

Deletion of the SELENOP gene leads to CNS atrophy with cerebellar ataxia in dogs.

We investigated a hereditary cerebellar ataxia in Belgian Shepherd dogs. Affected dogs developed uncoordinated movements and intention tremor at two weeks of age. The severity of clinical signs was highly variable. Histopathology demonstrated atrophy of the CNS, particularly in the cerebellum. Combined linkage and homozygosity mapping in a family with four affected puppies delineated a 52 Mb critical interval. The comparison of whole genome sequence data of one affected dog to 735 control genomes revealed a private homozygous structural variant in the critical interval, chr4:66,946,539_66,963,863del17,325. This deletion includes the entire protein coding sequence of SELENOP and is predicted to result in complete absence of the encoded selenoprotein P required for selenium transport into the CNS. Genotypes at the deletion showed the expected co-segregation with the phenotype in the investigated family. Total selenium levels in the blood of homozygous mutant puppies of the investigated litter were reduced to about 30% of the value of a homozygous wildtype littermate. Genotyping >600 Belgian Shepherd dogs revealed an additional homozygous mutant dog. This dog also suffered from pronounced ataxia, but reached an age of 10 years. Selenop-/- knock-out mice were reported to develop ataxia, but their histopathological changes were less severe than in the investigated dogs. Our results demonstrate that deletion of the SELENOP gene in dogs cause a defect in selenium transport associated with CNS atrophy and cerebellar ataxia (CACA). The affected dogs represent a valuable spontaneous animal model to gain further insights into the pathophysiological consequences of CNS selenium deficiency