Lokalisierung und Charakterisierung der AAA+-Proteine AFG1L2 und AFG1L1 aus Arabidopsis thaliana

Das Kalzium Signalnetzwerk ist ein wichtiger Übermittler von internen und externen Reizen in der Pflanze. Diese Signale werden unter anderem durch Calmodulin und calmodulin ähnlichen Proteine an verschiedene Effektorproteine weitergeleitet. AFG1L2 (AFG1 like Protein 2) wurde in dieser Arbeit als calmodulin bindendes Proteine aus der Familie der AAA+ Proteine (ATPasen associated with various cellular activities) identifiziert. Mit Hilfe von GFP Fusionskonstrukten konnte die in vivo Lokalisierung von AFG1L2 in Mitochondrien gezeigt werden, und die duale Lokalisierung des bereits zuvor beschriebenen Homologs AFG1L1 in Chloroplasten und Mitochondrien bestätigt werden. Die Bindung von Calmodulin erfolgt bei AFG1L2 kalziumabhängig und die Calmodulin Bindedomäne befindet sich homolog zu AFG1L1 nahe dem für die ATP Hydrolyse essenziellen Walker A Motiv in der AAA Domäne. Es konnte gezeigt werden, dass ADP die Interaktion von AFG1L2 und Calmodulin verstärkt. Das weist auf einen inhibierenden Effekt von Calmodulin hin, da die Nucleotid Bindetasche besetzt bleibt und keine erneute ATP Hydrolyse stattfinden kann. Neben den konservierten Motiven haben AAA+ Proteine die Bildung von Oligomeren, meist Hexameren gemeinsam. In der Regel ist der Komplex die hydrolytisch aktive Form. Es konnte gezeigt werden, dass AFG1L2 prinzipiell in der Lage ist Oligomere zu bilden, aber dass das Vorliegen als Komplex wahrscheinlich nicht für die Hydrolyse von ATP erforderlich ist. Um die Funktion von AFG1L1 und AFG1L2 in der Pflanze zu bestimmen, wurden afg1l1 und afg1l2 Mutanten untersucht. Diese zeigten aber unter den normalen Bedingungen keinen Phänotyp. Auch eine im Zuge dieser Arbeit generierte afg1l1/afg1l2 Doppel Mutante wies unter Standart-Bedingungen keine Beeinträchtigungen im Wachstum auf. Analysen der Expression der Proteine legen nahe, dass AFG1L1 und AFG1L2 unter Gewächshaus Bedingungen einander nicht substituieren. DNA Microarray Daten lassen jedoch auf eine Beteiligung von AFG1L1 an der Stress Toleranz gegen Salz und Hyper-Osmolarität schließen.The calcium-signaling network is an important transducer of internal and external stimuli in plants. These signals are transduced by a divers set of calcium-sensors such as calmodulin and calmodulin like proteins. In this work, AFG1L2 (AFG1 like protein 2) was characterized as a calmodulin binding protein that belongs to the family of AAA+ proteins (ATPases associated with various cellular activities). Using GFP fusion constructs it was possible to determine the in vivo localization of AFG1L2 in mitochondria and also to confirm the dual localization of a previously described homologe, AFG1L1, to mitochondria and chloroplasts. The interaction between AFG1L2 and calmodulin is calcium dependent and the calmodulin binding site of the AFG1L2 protein is in the AAA domain at a site homologous to the AFG1L1 protein, in very close proximity to the Walker A Motif, which is essential for ATP hydrolysis. It could be shown that ATP and ADP intensify the interaction between AFG1L2 and calmodulin. AAA-proteins are not only described by their conserved motifs but also tend to form oligomers, often hexameres. Usually, only the complex form is the hydrolytically active form. It was shown that AFG1L2 basically is able to form oligomers but the AFG1L2 monomer seems to be able to hydrolyze ATP. To determine the function of AFG1L1 and AFG1L2 in plants afg1l1 and afg1l2 mutant plants of Arabidopsis thaliana were analysed. Those mutant plants did not show any visible phenotype under normal growth conditions. Therefore a afg1l1/afg1l2 double mutant was generated but it also showed no visible phenotype. Analyses of the expression profile of AFG1L1 and AFG1L2 suggest that both proteins do not substitute for each other under non-stress conditions. But DNA Microarray data propose an involvement of AFG1L1 in salt stress tolerance or in tolerance against hyper-osmolarity

A Global Estimate of the Size and Location of Informal Settlements

Slums are a structural feature of urbanization, and shifting urbanization trends underline their significance for the cities of tomorrow. Despite their importance, data and knowledge on slums are very limited. In consideration of the current data landscape, it is not possible to answer one of the most essential questions: Where are slums located? The goal of this study is to provide a more nuanced understanding of the geography of slums and their growth trajectories. The methods rely on the combination of different datasets (city-level slum maps, world cities, global human settlements layer, Atlas of Informality). Slum data from city-level maps form the backbone of this research and are made compatible by differentiating between the municipal area, the urbanized area, and the area beyond. This study quantifies the location of slums in 30 cities, and our findings show that only half of all slums are located within the administrative borders of cities. Spatial growth has also shifted outwards. However, this phenomenon is very different in different regions of the world; the municipality captures less than half of all slums in Africa and the Middle East but almost two-thirds of all slums in cities of South Asia. These insights are used to estimate land requirements within the Sustainable Development Goals time frame. In 2015, almost one billion slum residents occupied a land area as large as twice the size of the country of Portugal. The estimated 380 million residents to be added up to 2030 will need land equivalent to the size of the country of Egypt. This land will be added to cities mainly outside their administrative borders. Insights are provided on how this land demand differs within cities and between world regions. Such novel insights are highly relevant to the policy actions needed to achieve Target 11.1 of the Sustainable Development Goals (“by 2030, ensure access for all to adequate, safe and affordable housing and basic services, and upgrade slums”) as interventions targeted at slums or informal settlements are strongly linked to political and administrative boundaries. More research is needed to draw attention to the urban expansion of cities and the role of slums and informal settlements

Responses to larval herbivory in the phenylpropanoid pathway of Ulmus minor are boosted by prior insect egg deposition

Main conclusion Elms, which have received insect eggs as a ‘warning’ of larval herbivory, enhance their anti-herbivore defences by accumulating salicylic acid and amplifying phenylpropanoid-related transcriptional and metabolic responses to hatching larvae. Abstract Plant responses to insect eggs can result in intensified defences against hatching larvae. In annual plants, this egg-mediated effect is known to be associated with changes in leaf phenylpropanoid levels. However, little is known about how trees—long-living, perennial plants—improve their egg-mediated, anti-herbivore defences. The role of phytohormones and the phenylpropanoid pathway in egg-primed anti-herbivore defences of a tree species has until now been left unexplored. Using targeted and untargeted metabolome analyses we studied how the phenylpropanoid pathway of Ulmus minor responds to egg-laying by the elm leaf beetle and subsequent larval feeding. We found that when compared to untreated leaves, kaempferol and quercetin concentrations increased in feeding-damaged leaves with prior egg deposition, but not in feeding-damaged leaves without eggs. PCR analyses revealed that prior insect egg deposition intensified feeding-induced expression of phenylalanine ammonia lyase (PAL), encoding the gateway enzyme of the phenylpropanoid pathway. Salicylic acid (SA) concentrations were higher in egg-treated, feeding-damaged leaves than in egg-free, feeding-damaged leaves, but SA levels did not increase in response to egg deposition alone—in contrast to observations made of Arabidopsis thaliana. Our results indicate that prior egg deposition induces a SA-mediated response in elms to feeding damage. Furthermore, egg deposition boosts phenylpropanoid biosynthesis in subsequently feeding-damaged leaves by enhanced PAL expression, which results in the accumulation of phenylpropanoid derivatives. As such, the elm tree shows similar, yet distinct, responses to insect eggs and larval feeding as the annual model plant A. thaliana

"Cause being sick doesn\u27t mean being worse" - student perspectives on bioethics concerning prenatal diagnostics

Bioethisches Lernen ist ein gleichermaßen notwendiges wie schwieriges Geschäft und stellt Lehrende wie Lernende vor besondere Herausforderungen. Angesichts der Komplexität des Gegenstands erfolgt Urteilsbildung als ein Ineinandergreifen von Einstellungen, Sachwissen und Kriterien. Vor diesem Hintergrund wurden im Rahmen eines empirischen Unterrichtsforschungsprojektes Prozesse bioethischen Lernens unter SchülerInnen der Sekundarstufe I untersucht. Im Zentrum der qualitativen Studie stand die Frage nach Konzepten und Kategorien bioethischen Urteilens zum Themenfeld Pränataldiagnostik. Der Beitrag stellt zunächst die identifizierten Kategoriensysteme vor, die einerseits die Frage nach dem Moralstatus menschlicher Embryonen und andererseits den Umgang mit Behinderung betreffen. Anhand der Kernkategorie werden die eruierten Kategorien in ein Zusammenhangsmodell überführt und im Blick auf das zentrale Phänomen diskutiert. (DIPF/Orig.)Bioethical learning is an essential as well as difficult topic that challenges both teachers and learners. Due to the complexity of the issue the formation of an opinion requires the combination of attitudes, factual knowledge and specific criteria. Against this background, processes of bioethical learning have been examined among students in grade 10 as part of an empirical research project on teaching. This qualitative study focused on concepts and categories of forming a bioethical opinion about prenatal diagnostics. This contribution will present the identified systems of categories, which on the one hand deal with the question of the moral status of human embryos and on the other hand consider dealing with handicaps. With the help of the main category the other determined categories will be transferred into a coherent model and will then be discussed with respect to the central phenomenon. (DIPF/Orig.

Responses to larval herbivory in the phenylpropanoid pathway of Ulmus minor are boosted by prior insect egg deposition

Plant responses to insect eggs can result in intensified defences against hatching larvae. In annual plants, this eggmediated effect is known to be associated with changes in leaf phenylpropanoid levels. However, little is known about how trees—long-living, perennial plants—improve their egg-mediated, anti-herbivore defences. The role of phytohormones and the phenylpropanoid pathway in egg-primed anti-herbivore defences of a tree species has until now been left unexplored. Using targeted and untargeted metabolome analyses we studied how the phenylpropanoid pathway of Ulmus minor responds to egg-laying by the elm leaf beetle and subsequent larval feeding. We found that when compared to untreated leaves, kaempferol and quercetin concentrations increased in feeding-damaged leaves with prior egg deposition, but not in feeding damaged leaves without eggs. PCR analyses revealed that prior insect egg deposition intensified feeding-induced expression of phenylalanine ammonia lyase (PAL), encoding the gateway enzyme of the phenylpropanoid pathway. Salicylic acid (SA) concentrations were higher in egg-treated, feeding-damaged leaves than in egg-free, feeding-damaged leaves, but SA levels did not increase in response to egg deposition alone—in contrast to observations made of Arabidopsis thaliana. Our results indicate that prior egg deposition induces a SA-mediated response in elms to feeding damage. Furthermore, egg deposition boosts phenylpropanoid biosynthesis in subsequently feeding-damaged leaves by enhanced PAL expression, which results in the accumulation of phenylpropanoid derivatives. As such, the elm tree shows similar, yet distinct, responses to insect eggs and larval feeding as the annual model plant A. thaliana.</p

Towards a comprehensive understanding of the structural dynamics of a bacterial diterpene synthase during catalysis

Terpenes constitute the largest and structurally most diverse natural product family. Most terpenoids exhibit a stereochemically complex macrocyclic core, which is generated by C–C bond forming of aliphatic oligo-prenyl precursors. This reaction is catalysed by terpene synthases (TPSs), which are capable of chaperoning highly reactive carbocation intermediates through an enzyme-specific reaction. Due to the instability of carbocation intermediates, the proteins’ structural dynamics and enzyme:substrate interactions during TPS catalysis remain elusive. Here, we present the structure of the diterpene synthase CotB2, in complex with an in crystallo cyclised abrupt reaction product and a substrate-derived diphosphate. We captured additional snapshots of the reaction to gain an overview of CotB2’s catalytic mechanism. To enhance insights into catalysis, structural information is augmented with multiscale molecular dynamic simulations. Our data represent fundamental TPS structure dynamics during catalysis, which ultimately enable rational engineering towards tailored terpene macrocycles that are inaccessible by conventional chemical synthesis

Satellite-Dominated Sulfur L2,3_{2,3} X-ray Emission of Alkaline Earth Metal Sulfides

The sulfur L2,3 X-ray emission spectra of the alkaline earth metal sulfides BeS, MgS, CaS, SrS, and BaS are investigated and compared with spectra calculations based on density functional theory. Very distinct spectral shapes are found for the different compounds. With decreasing electronegativity of the cation, that is, increasing ionic bonding character, the upper valence band width and its relative spectral intensity decrease. These general trends are qualitatively reproduced by the spectra calculations, which give quite an accurate description of the spectral shapes in the upper valence band region. On the low energy side of the sulfur 3s → 2p transition dominating the spectra, we find strong satellites caused by “semi-Auger” decays involving configuration interaction. These satellites, previously believed to be energetically forbidden for sulfur L2,3 emission and only observed for the L2,3 emission of Cl to Cr, increase in intensity as the bonding character becomes more ionic and dominate the spectra for SrS and BaS. The intensities, energies, and widths of the satellites vary strongly between the investigated compounds, giving a very specific spectral fingerprint that can be used for speciation analysis

Laparoscopic adrenalectomy for functioning and non-functioning adrenal tumours

Background: The purpose of this study was a retrospective analysis of outcomes following laparoscopic adrenalectomy (LA) performed for benign adrenal tumours responsible for various endocrinological disorders. The patients were diagnosed with non-functioning (NFT) and functioning adrenal tumours (FT) including pheochromocytoma (PH), Conn&#8217;s syndrome (CO) and Cushing&#8217;s (CS) syndrome. Material and methods: A total of 165 LAs were carried out between August 1995 and September 2009 via either the transperitoneal (n = 38) or retroperitoneal (n = 127) approach. The analysed factors included demographic data of patients, the American Association of Anaesthesiology score (ASA), indication for surgery, tumour size and side, intraoperative and postoperative outcome of LA including duration of surgery, blood loss, time until ambulation, length of hospital stay, time until return to normal activity, the complication rate, as well as the conversion rate to open adrenalectomy. Results: There were 111 patients with NFT and 54 with FT. Patients with NFT were significantly older than those with CO (p < 0.05). The mean size of the lesion differed between CO and other adrenal tumours (p < 0.05) as well as between NFT and PH (p < 0.05). All the lesions except aldosteronomas were detected predominantly in the right adrenal gland (p < 0.05). However, despite the different characteristic and clinical disorders related to laparoscopically removed adrenal tumours, the intraoperative and postoperative outcomes did not significantly differ in most cases between the analysed groups of patients. Conclusion: This study shows that LA is a safe, effective, and well-tolerated procedure despite the hormonal activity of the removed lesions. Minimal invasive surgery may be recommended as the &#8216;gold standard&#8217; in the treatment of both functioning and non-functioning benign tumours of the adrenal gland. (Pol J Endocrinol 2011; 62 (6): 512&#8211;516)Wstęp: Celem tego badania była retrospektywna analiza wyników adrenalektomii laparoskopowej (LA) wykonywanej z powodu łagodnych guzów nadnerczy mogących powodować różne zaburzenia endokrynologiczne. U pacjentów rozpoznawano nieczynne hormonalnie guzy nadnerczy (NFT) oraz hormonalnie czynne guzy nadnerczy (FT), w tym guz chromochłonny (PH), zespół Conna (CO) i zespół Cushinga (CS). Materiał i metody: Pomiędzy sierpniem 1995 r. a wrześniem 2009 r. wykonano łącznie 165 LA z dostępu przezotrzewnowego (n = 38) lub zaotrzewnowego (n = 127). Analizowanymi czynnikami były: dane demograficzne pacjentów, wynik skali ASA, wskazanie do operacji, rozmiar guza i jego umiejscowienie, śródoperacyjne i pooperacyjne wyniki LA w tym czas trwania operacji, utratę krwi, czas uruchomienia chorych, długość hospitalizacji, czas powrotu do normalnej aktywności, śródoperacyjne i pooperacyjne powikłania, jak również wskaźnik konwersji do otwartej adrenalektomii. Wyniki: Badaniem objęto 111 pacjentów z NFT i 54 z FT. Pacjenci z NFT byli istotnie starsi niż pacjenci z CO (p < 0,05). Średnia wielkość zmiany różniła się pomiędzy CO i innymi guzami nadnerczy (p < 0,05), jak również pomiędzy NFT i PH (p < 0,05). Wszystkie zmiany za wyjątkiem aldosteronoma wykrywano głównie w prawym nadnerczu (p < 0,05). Mimo różnych cech charakterystycznych oraz zaburzeń klinicznych związanych z usuwanymi laparoskopowo guzami nadnerczy w większości przypadków nie było istotnych różnic wyników środoperacyjnych i pooperacyjnych pomiędzy analizowanymi grupami pacjentów. Wnioski: Przedstawione badanie pokazuje, że LA jest bezpiecznym, efektywnym i dobrze tolerowanym przez chorych zabiegiem mimo czynności hormonalnej usuwanych zmian ogniskowych. Małoinwazyjny zabieg operacyjny można uznać za metodę referencyjną w chirurgii zarówno czynnych, jak i nieczynnych hormonalnie łagodnych guzów nadnerczy. (Endokrynol Pol 2011; 62 (6): 512&#8211;516