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The pi-calculus is a well-analyzed model for mobile processes and mobile computations.
While a lot of other process and lambda calculi that are core languages of higher-order concurrent and/or functional programming languages use a contextual semantics observing the termination behavior of programs in all program contexts, traditional program equivalences in the pi-calculus are bisimulations and barbed testing equivalences, which observe the communication capabilities of processes under reduction and in contexts.
There is a distance between these two approaches to program equivalence which makes it hard to compare the pi-calculus with other languages. In this paper we contribute to bridging this gap by investigating a contextual semantics of the synchronous pi-calculus with replication and without sums.
To transfer contextual equivalence to the pi-calculus we add a process Stop as constant which indicates success and is used as the base to define and analyze the contextual equivalence which observes may- and should-convergence of processes.
We show as a main result that contextual equivalence in the pi-calculus with Stop conservatively extends barbed testing equivalence in the (Stop-free) pi-calculus. This implies that results on contextual equivalence can be directly transferred to the (Stop-free) pi-calculus with barbed testing equivalence.
We analyze the contextual ordering, prove some nontrivial process equivalences, and provide proof tools for showing contextual equivalences. Among them are a context lemma, and new notions of sound applicative similarities for may- and should-convergence.
The Paddys River Wetlands in the New South Wales Southern Highlands, southwest of Sydney, are characterised by several watercourses with associated swamps (fens), some of which, on Forestry Corporation of NSW land, have been the focus for removal of Pinus radiata wildings by the Penrose Swamps Conservation Group. In this study we map a population of Eucalyptus aquatica trees in one of these swamps perched above Paddys River (latitude 34.65575o S, longitude 150.21831o E; 600 m elevation). Eucalyptus aquatica is geographically restricted to the Paddys River area and is listed as a threatened species at state and national levels. New findings on the physical characteristics of the swamp in relation to the bedrock geology, stream geomorphology, peat development and the main native plant species, are presented. The occurrence of clumps of Eucalyptus aquatica appears to be independent of the type or thickness of the growing substrate. Rather it is suggested that a continuous supply of water and the shelter afforded by the narrow valley may be key factors determining the distribution of the trees at the study site. An on-going programme of research is underway to study other occurrences of Eucalyptus aquatica.
The Queensland Herbarium Regional Ecosystem Survey and Mapping (QHRESM) program has contributed almost 90000 (89389) specimens to the Queensland Herbarium in Brisbane accounting for 28% of the specimens added to the Herbarium between 1970 and 2011. These specimens have been collected across all bioregions and vegetation communities in Queensland in a systematic sampling program driven by the requirement to sample comprehensively all vegetation communities. The QHRESM’s Queensland Herbarium (BRI) specimens represent more than 79% of the native, and 73% of the naturalised vascular flora of Queensland, as well as making valuable contributions to the bryophytes, lichens and liverworts collections. The data and specimens collected enhance our ability to assess local, state and continental-scale plant diversity, and will be used by botanists, ecologists, governments, business and the public for long into the future.
Natural vegetation of freely draining beach sand ridges in the valley of the Upper Myall River on the lower North Coast of NSW is sclerophyllous open forest or woodland. Based on previous experience on the nearby Eurunderee area, the vegetation on the sand ridges was classified into three types. These were mapped using aerial photographs and field observation. In transects, each 50 m long, the presence of species was scored in 10, 5 X 5 m quadrats. 48 such transects were used sampling all three types of vegetation on the sand ridges and also vegetation in periodically waterlogged sites adjacent to the ridges. Data from the transects, subjected to an ordination using principal components analysis, revealed clear separation between vegetation of the ridges and that of periodically waterlogged sites. In the ordination, vegetation of the ridges formed a continuum with the three types occupying characteristic parts of the continuum, reflecting their respective distributions on sands with different geomorphological histories. The most grassy, tallest forest, termed Dry Sclerophyll Forest (DSF) is on sands either recently disturbed or deposited (Holocene) or closely overlying other substrates. Banksia serrata occurs in DSF. The least grassy, most sclerophyllous, lowest forest or woodland, termed Dry Heath Forest (DHF), occurs on sands apparently little disturbed since they were laid down in the Pleistocene. Banksia aemula occurs in DHF. An intermediate forest, in which Banksia aemula and Banksia serrata occur together, Intermediate Dry Forest (IDF), is most widely found on the sand mass close to Bombah Broadwater. This sand is postulated to have been reworked during the last Glacial Period. In short, the vegetation of these sand ridges largely varies with time since they were laid down or last disturbed in a major way. Preliminary observations indicate the degree of podsolization of their soils is similarly related to this variation in time.
Lysimachia mauritiana Lam. (family Primulaceae), a small short-lived herb native to India, Indian and Pacific Ocean islands, and coastal east Asia, is described as a new naturalised record from the eastern suburbs of Sydney, New South Wales, Australia. It was first recorded in 1981 near Coogee, and grows in exposed rock crevices and seepages on the seacoast, very similar to its natural habitat overseas. Lysimachia mauritiana is known to have been cultivated in the area in 1961 in a home garden, which is the likely source of this introduction; it appears to be spreading locally as a weed.
Vegetation of Little Bora Nature Conservation Trust Agreement, North Western Slopes, New South Wales
(2014)
The vegetation of the Little Bora Nature Conservation Trust Agreement property (560 ha in area), 8 km south east of Bingara (lat 29° 55’S long 150° 37’) in the Gwydir Shire and within the Nandewar Bioregion is described. Eight vegetation communities are defined based on flexible UPGMA analysis of cover-abundance scores of all vascular plant taxa. These communities are mapped based on ground truthing, ADS40 imagery interpretation, topography and substrate. Communities described are: 1) Melaleuca bracteata – Eucalyptus melanophloia – Eucalyptus camaldulensis Woodland, 2) Callitris glaucophylla – Eucalyptus melanophloia – Eucalyptus albens Woodland, 3) Callitris glaucophylla – Eucalyptus melanophloia – Brachychiton populneus Woodland, 4) Eucalyptus albens Woodland, 5) Eucalyptus caleyi – Eucalyptus albens – Callitris glaucophylla Woodland, 6) Callitris glaucophylla – Eucalyptus melanophloia – Eucalyptus albens Woodland, 7) Austrostipa verticillata – Austrostipa scabra Derived Grassland, 8) Eucalyptus melliodora – Eucalyptus dealbata Woodland. A total of 232 vascular plant taxa were found of which 14% were considered exotic in origin. 66 ha of listed threatened communities were mapped along with populations of a currently listed Extinct plant (TSC Act) Dodonaea stenophylla.
A population of 700 Blandfordia cunninghamii Lindl. (family Blandfordiaceae) plants in the Blue Mountains, 100 km west of Sydney, New South Wales was monitored over a period of seven years, during which a part of the population area was burnt in a Hazard Reduction Burn (HRB). The survey measured flowering of Blandfordia cunninghamii in both the burnt and unburnt areas. In part of the unburnt area flowering (in December) was strongly correlated with previous September rainfall, but in another unburnt area there was no flowering at all over the seven years. An enhanced flowering response after fire was found in the burnt area and the diminution of this enhanced response in subsequent years was found to be logarithmic (taking into account potential rainfall effects). No recruitment of juvenile plants after fire was observed. 87% of seeds of Blandfordia cunninghamii were found to be germinable. Slow juvenile growth of Blandfordia cunninghamii in the field was measured over seven years. Seed was collected for two major seedbanks, the NSW Plantbank at the Australian Botanic Garden, Mt Annan and the Millennium Seedbank at Kew in the United Kingdom.
Background: Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls.
Principal findings: In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4×10−6) and 14 (IGHV1-67 p = 7.9×10−8) which indexed novel susceptibility loci.
Significance: The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.
Der vollständige Titel der vorliegenden Arbeit lautet: Sprachwandel in Chur: 'Aufnahmen des Sprachatlasses der deutschen Schweiz (SDS) konfrontiert mit der Mundart von heute'. Entsprechend dieses Programmes sollen folgende Fragen beantwortet werden:
o Was für Aenderungen können wir in der Churer Mundart ausmachen?
o In welche Richtung gehen diese Aenderungen? Lassen sich Tendenzen ausmachen?
o Welche Einflüsse führten zu den festgestellten Veränderungen?
Es ist klar, dass im Rahmen dieser Arbeit nur ein Teil allen Sprachwandels in Chur festgehalten werden konnte. Es ist aber durchaus möglich, anhand der erfassen Veränderungen Schlüsse zu ziehen, die sich auch auf die Mundart von Chur überhaupt übertragen lassen.
Schwerpunktmässig wurden für diese Arbeit Vokalismus und morphologisch-syntaktische Probleme bevorzugt behandelt. Für Konsonantismus eignet sich die Form der schriftlichen Umfrage wenig (Vgl. Kap.l.3.). Der Wandel im mundartlichen Wortschatz ist zu gross, als dass er auch nur annähernd vollständig behandelt werden könnte. Und Stiefkinder mussten auch satzmelodische und rhythmische Probleme bleiben. Dafür wurden anhand ausgesuchter Beispiele Phänomene erfasst, bei welchen der SDS nicht als Grundlage dienen konnte.
Wichtig ist für diese Arbeit, dass mit "der Mundart von heute" auch wirklich heutige Mundart erfasst wurde. Es sollte also keineswegs eine "richtige" Churer Mundart rekonstruiert (auch wenn dies mit den Verweisen auf die SDS-Karten implizit natürlich gemacht worden ist), sondern vielmehr mit den Aussagen der Gewährspersonen (Gwp) gearbeitet werden. In diesem Sinne kann Sekundärliteratur Erhellung bringen, soll aber nicht Untersuchungsgegenstand sein.
SAFE Newsletter : 2014, Q2
(2014)
Many studies about endocrine pollution in the aquatic environment reveal changes in the reproduction system of biota. We analysed endocrine activities in two rivers in Southern Germany using three approaches: (1) chemical analyses, (2) in vitro bioassays, and (3) in vivo investigations in fish and snails. Chemical analyses were based on gas chromatography coupled with mass spectrometry. For in vitro analyses of endocrine potentials in water, sediment, and waste water samples, we used the E-screen assay (human breast cancer cells MCF-7) and reporter gene assays (human cell line HeLa-9903 and MDA-kb2). In addition, we performed reproduction tests with the freshwater mudsnail Potamopyrgus antipodarum to analyse water and sediment samples. We exposed juvenile brown trout (Salmo trutta f. fario) to water downstream of a wastewater outfall (Schussen River) or to water from a reference site (Argen River) to investigate the vitellogenin production. Furthermore, two feral fish species, chub (Leuciscus cephalus) and spirlin (Alburnoides bipunctatus), were caught in both rivers to determine their gonadal maturity and the gonadosomatic index. Chemical analyses provided only little information about endocrine active substances, whereas the in vitro assays revealed endocrine potentials in most of the samples. In addition to endocrine potentials, we also observed toxic potentials (E-screen/reproduction test) in waste water samples, which could interfere with and camouflage endocrine effects. The results of our in vivo tests were mostly in line with the results of the in vitro assays and revealed a consistent reproduction-disrupting (reproduction tests) and an occasional endocrine action (vitellogenin levels) in both investigated rivers, with more pronounced effects for the Schussen river (e.g. a lower gonadosomatic index). We were able to show that biological in vitro assays for endocrine potentials in natural stream water reasonably reflect reproduction and endocrine disruption observed in snails and field-exposed fish, respectively.
We study consumption-portfolio and asset pricing frameworks with recursive preferences and unspanned risk. We show that in both cases, portfolio choice and asset pricing, the value function of the investor/representative agent can be characterized by a specific semilinear partial differential equation. To date, the solution to this equation has mostly been approximated by Campbell-Shiller techniques, without addressing general issues of existence and uniqueness. We develop a novel approach that rigorously constructs the solution by a fixed point argument. We prove that under regularity conditions a solution exists and establish a fast and accurate numerical method to solve consumption-portfolio and asset pricing problems with recursive preferences and unspanned risk. Our setting is not restricted to affine asset price dynamics. Numerical examples illustrate our approach.
We study self- and cross-excitation of shocks in the Eurozone sovereign CDS market. We adopt a multivariate setting with credit default intensities driven by mutually exciting jump processes, to capture the salient features observed in the data, in particular, the clustering of high default probabilities both in time (over days) and in space (across countries). The feedback between jump events and the intensity of these jumps is the key element of the model. We derive closed-form formulae for CDS prices, and estimate the model by matching theoretical prices to their empirical counterparts. We find evidence of self-excitation and asymmetric cross-excitation. Using impulse-response analysis, we assess the impact of shocks and a potential policy intervention not just on a single country under scrutiny but also, through the effect on cross-excitation risk which generates systemic sovereign risk, on other interconnected countries.
Exit strategies
(2014)
We study alternative scenarios for exiting the post-crisis fiscal and monetary accommodation using a macromodel where banks choose their capital structure and are subject to runs. Under a Taylor rule, the post-crisis interest rate hits the zero lower bound (ZLB) and remains there for several years. In that condition, pre-announced and fast fiscal consolidations dominate - based on output and inflation performance and bank stability - alternative strategies incorporating various degrees of gradualism and surprise. We also examine an alternative monetary strategy in which the interest rate does not reach the ZLB; the benefits from fiscal consolidation persist, but are more nuanced.
We study the behavioral underpinnings of adopting cash versus electronic payments in retail transactions. A novel theoretical and experimental framework is developed to primarily assess the impact of sellers’ service fees and buyers’ rewards from using electronic payments. Buyers and sellers face a coordination problem, independently choosing a payment method before trading. In the experiment, sellers readily adopt electronic payments but buyers do not. Eliminating service fees or introducing rewards significantly boosts the adoption of electronic payments. Hence, buyers’ incentives play a pivotal role in the diffusion of electronic payments but monetary incentives cannot fully explain their adoption choices. Findings from this experiment complement empirical findings based on surveys and field data.
In dieser Arbeit wurde der langsame Neutroneneinfang (s-Prozess) mit dem Nukleosynthese-Programm NETZ simuliert. Ziel solcher Programme ist es, die solare Häufigkeitsverteilung zu reproduzieren.
Der s-Prozess dient der Synthese von Elementen schwerer als Eisen und ereignet sich in astrophysikalischen Szenarien mit relativ geringen Neutronendichten. Dadurch sind die Neutroneneinfangzeiten meist größer als die Betazerfallszeiten und der Prozesspfad folgt dem Stabilitätstal in der Nuklidkarte. Aus diesem Grund sind die Reaktionsraten gut messbar und es steht ein umfangreiches Daten-Netzwerk zur Verfügung, welches in die Simulationen einfließen kann.
Man unterschiedet zwischen der schwachen- und der Hauptkomponente des s-Prozesses. Die schwache Komponente findet in massereichen Sternen (M > 8M⊙) beim Helium-Kernbrennen und Kohlenstoff-Schalenbrennen statt. Bei Temperaturen über 2.5 × 108 K wird die Reaktion 22Ne(α ,n)25Mg aktiviert, welche Neutronen liefert, die von der Eisensaat eingefangen werden. Bei einer mittleren Neutronendichte von 106/cm3 reicht die Neutronenbestrahlung jedoch nicht aus, um den Synthesefluss über die abgeschlossene Neutronenschale bei N = 50 hinweg zu treiben. Folglich werden nur Isotope zwischen Eisen und Yttrium (56 < A < 90) aufgebaut.
Schwerere Isotope (90 ≤ A ≤ 208) werden dagegen in der Hauptkomponente synthetisiert. Diese findet in thermisch pulsierenden AGB-Sternen statt, in denen während des Helium-Schalenbrennens Neutronen hauptsächlich über die Reaktion 13C(α ,n)16O zur Verfügung gestellt werden.
Am Ende der jeweiligen Brennphasen gibt es einen Anstieg von Temperatur und Neutronendichte, welche jedoch nicht die globale Häufigkeitsverteilung, wohl aber Verzweigungspunkte beeinflussen können. An diesen Punkten liegen die Neutroneneinfang- und Betazerfallszeiten in der gleichen Größenordnung, sodass der s-Prozesspfad aufspaltet.
Hinzu kommt, dass unter stellaren Bedingungen die Reaktionsraten starken Änderungen unterworfen sein können. Bei hohen Temperaturen und Dichten befinden sich die Kerne in angeregten Zuständen, die wie auch der Grundzustand Neutronen einfangen oder radioaktiv zerfallen können, jedoch bei veränderten Raten. Dieser Sachverhalt kann einen Einfluss auf die Häufigkeitsverteilung haben.
Das umfangreiche Reaktionsnetzwerk des s-Prozesses kann schnell und mit guter Genauigkeit mit dem Programm NETZ berechnet werden. Dabei muss dem Programm ein Neutronenpuls - der zeitliche Verlauf von Neutronendichte und Temperatur - vorgegeben werden. Ziel dieser Arbeit war es, einen geeigneten solchen Puls zu finden, um die bisherigen Ergebnisse von NETZ zu optimieren. Außerdem wurde eine Aktualisierung der Reaktionsraten und solaren Häufigkeitsverteilung durchgeführt.
Die neuen Neutronenpulse für die schwache- und Hauptkomponente liefern eine Verbesserung in der Übereinstimmung von berechneter und solarer Häufigkeit. Dabei konnte für die Hauptkomponente sowohl ein Profil mit einem rechteckigen als auch mit einem exponentiellen Verlauf der Neutronendichte gefunden werden.
Darüber hinaus bietet NETZ die Möglichkeit, den Einfluss veränderter Reaktionsraten auf die Häufigkeitsverteilung abzuschätzen. Dazu steht inzwischen auch ein Online-Interface zur Verfügung. Dies ist besonders interessant, wenn es neue Messungen z.B. für Neutroneneinfangreaktionen gibt und man die Relevanz für den s-Prozess bestimmen möchte. So konnte in dieser Arbeit die Bedeutung der kürzlich neu gemessenen Raten für 63,65Cu(n,γ) und 69,71Ga(n,γ) beurteilt werden.
Alzheimer’s disease (AD) is a common, age associated neurodegenerative disease that manifests as progressive dementia and is characterized by accumulation of the amyloid beta (Aβ) peptide which is a processing product of a transmembrane protein termed Alzheimer Amyloid Precursor Protein (APP). The Aβ peptide is generated by a sequential proteolytic processing of APP by two distinct proteases that are termed β- and γ-secretase. The β-secretase, also called BACE-1 or memapsin 2, belongs to the family of aspartyl proteases. BACE-1 evidently cleaves APP in an acidic endosomal compartment after endocytosis of APP, thereby facilitating Aβ peptide generation.
Sorting of transmembrane proteins is generally controlled by sorting signals in the cytoplasmic domains of the cargo proteins. The short cytoplasmic tail of BACE-1 with 23 amino acids contains a sorting signal of the acidic cluster, di-leucine (ACDL) type. The two Leu residues in this determinant are important for the clathrin mediated endocytosis of BACE-1, whereas the acidic residues together with the Leu are required for the endosomal sorting and recycling of BACE-1 back to the plasma membrane. The ACDL motif binds to the members of the GGA (Golgi-localized γ ear-containg ARF- binding proteins) family (GGA1-GGA3) that are involved in the sorting of BACE-1.
One of the major aims of this study was to address the role of flotillins in the intracellular sorting of BACE-1. This study shows that flotillin-1 directly binds to the di-leucine motif in the cytoplasmic tail of BACE-1, whereas flotillin-2 only shows an association mediated by flotillin-1. Flotillin-1 competes with GGA2 for the binding to BACE-1 tail, and thus influences the endosomal sorting of BACE-1. Importantly, depletion of flotillins results in an altered localization of the wildtype BACE-1, whereas the plasma membrane resident Leu to Ala (LLAA) mutant is not affected. Flotillin knockdown results in an accumulation of BACE-1, implicating reduced degradation and enhanced stability of this protease. Thus, flotillins appear to be important for the cellular targeting of BACE-1 and also influence the amyloidogenic processing of APP, as demonstrated by an increase in the amyloidogenic C-99 processing fragments.
When flotillin depleted cells were subjected to apoptotic stresses including Aβ25-35 synthetic peptide (inducer of the extrinsic apoptosis pathway) or several chemotherapeutic agents (staurosporine, brefeldin A, doxorubicin, carboplatin and paclitaxel: intrinsic apoptosis pathway) and cytotoxicity was determined, various apoptotic markers were activated in flotillin depleted cells. Caspase-3 and GGA3 are well accepted apoptosis markers and an enhanced caspase-3 cleavage was detected upon STS induced apoptosis in SH-SY5Y, HeLa, and HaCaT cell lines and increased GGA3 cleavage was observed in MCF7 cell line.
One of the major reasons for the apoptotic sensitivity in the absence of flotillins was a PI3K/Akt signaling defect. Neuroblastoma cells depleted of flotillins showed diminished levels of total Akt, phospho-Akt and phospho-ERK upon STS induced apoptosis. Since PI3K/Akt was the primary survival pathway affected upon STS induced apoptosis, ectopic expression of Akt in neuroblastoma cell line reduced caspase-3 cleavage and retarded apoptosis.
The direct downstream target of Akt is FOXO3a, whose localization was investigated in flotillin depleted cells. A major proportion of FOXO3a was localized in the nucleus of flotillin knockdown cells, implicating that FOXOs are active in these cells and subsequently trigger the transcription of death genes. Strikingly, an essential anti-apoptotic molecule and a major cancer target, Mcl-1, was inherently downregulated in flotillin knockdown cells. Mcl-1 is a chief member of the Bcl-2 family as it plays a pivotal role in cell survival and it is a critical protein in cancer therapeutics as suppression of Mcl-1 protein can curtail the survival and growth of tumorous cells.
Neuroblastoma cells were rescued from undergoing permanent damage due to STS induced apoptosis by overexpression of anti-apoptotic Bcl-2. Phorbol esters are well known PKC activators, and pre-treatment of neuroblastoma cells with phorbol esters along with staurosporine reduced caspase-3 cleavage.
These results demonstrate that absence of flotillins can sensitize cellular systems to apoptosis induction. The two main characteristics of cancer cells include resistance to apoptosis and unresponsiveness to chemotherapeutic agents. It is a well established fact that impaired apoptosis is central to tumour development. This study implicates that the downregulation of flotillin function can trigger cellular susceptibility and enhances apoptosis in response to conventional chemotherapeutic agents. Therefore, flotillins can serve as vital regulators in providing a more rational approach in molecular-targeted therapies for receding cancer growth and survival.
5-lipoxygenase (5-LO) is an enzyme with a substantial role in inflammatory processes. In vitro kinase assays using [32P]-ATP in combination with mutagenesis have revealed that serine residues 271, 523 and 663 can be phosphorylated by MK2, PKA and ERK2 kinases, respectively. A few available reports regarding 5-LO protein sequence have covered up to 30% of the sequence after amino acid sequencing including Ser663. In LCMS/MS analyses of 5-LO tryptic digests from different cellular sources different peptides have been detected; however, none of the three phosphorylations has been detected and only Ser663 was included in the covered sequence.
As there was no comprehensive mass spectrometric analysis of 5-LO, the purpose of this study was to optimize the experimental conditions under which detection of the aforementioned phosphorylation events, as well as other possible post-translational modifications (PTMs), would be feasible. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was used for peptide analysis of 5-LO cleaved either by chemical reagents or by proteases. Sequence coverage of 5-LO could be enhanced to be close to completion by combination of results from digestions by trypsin, AspN and chymotrypsin. In-gel trypsin digestion followed by in-solution AspN digestion proved to be a useful sample treatment for reproducible detection of the Ser271-containing peptide.
Nevertheless, in none of the examined cleavage protocols the sequence around Ser523 was detected reproducibly or with acceptable signal intensity for subsequent peptide fragmentation. Propionic anhydride and sulfo-NHS-SS-biotin cross-linker (EZ-linkTM), were used for derivatization of lysine side chains and hindrance of lysine residue recognition by trypsin. Phosphopeptide enrichment became possible after tryptic digestion of these samples, not only due to formation of an individual Ser523-containing peptide, but also because TiO2-mediated enrichment, which is performed in acidic pH, was not impaired by positively charged free lysine side chains. Additionally, biotinylation of lysine residues was exploited for an intermediate enrichment step of the lysine containing peptides, prior to TiO2 phosphopeptide enrichment.
MALDI-MS analysis after in-vitro phosphorylation of 5-LO by the three kinases showed that Ser271 was phosphorylated in the MK2 and PKA kinase assays, while Ser523 was phosphorylated only in the PKA kinase assay. Surpisingly, no phosphopeptides were detected in the in-vitro kinase assays with ERK2, even though the unmodified counterpart of the Ser663-containing peptide was easily detected. The detection limit for each of the three phosphorylation sites was determined by the use of custom made phosphopeptides and an amount of 0.06 pmol of phosphopeptide in 1 μg 5-LO (representing 0.5% phosphorylation rate) was sufficient in all cases for successful enrichment and detection by MS.
In-vitro kinase assays with [32P]-ATP were performed for some kinases that were expected to phosphorylate 5-LO according to in-silico data. Three members of the Src tyrosine kinase family (Fgr, Hck and Yes) and the Ser/Thr specific kinase DNA-PK used 5-LO as their substrate and mainly residues at the N-terminal part of 5-LO were detected phosphorylated by MS (e.g. Y42, Y53). Additional in-vitro assays for recombinant 5-LO modification included incubation with glutathione or compound U73122, previously described as inhibitor of 5-LO.
Since in-vitro assays might have generated artifacts, a method for 5-LO purification from human cells was sought, in order to examine the modification state of the protein in the cellular context. ATP-agarose affinity purification and anti-5-LO immunoprecipitation proved inappropriate for sample purification for MALDI-MS analysis. Consequently, two human cell lines that are able to express 5-LO (Rec-1 Blymphocytes and MM6 monocytes) were transduced with a DNA cassette that contained recombinant human 5-LO sequence with an attached N-terminal FLAG-tag. Anti-FLAG immunoprecipitation was then performed effectively in cell lysates and the precipitated FLAG-5-LO was separated by SDS-PAGE before MALDI-MS analysis.
The examined cell stimuli were expected to result to phosphorylation of 5-LO at Ser523 by PKA in Rec-1 cells and to phosphorylation of Ser271 and/or Ser663 in MM6 cells by activated MK2 and ERK2, respectively. Additionally, under the conditions of MM6 cell stimulation, Fgr, Hck and Yes kinases, which phosphorylated 5-LO in vitro, were expected to be activated and the possibility of 5-LO phosphorylation on tyrosine was investigated. Although immunoblotting results indicated that all the aforementioned phosphorylation events existed in the examined samples, MALDI-MS analysis verified only phosphorylation on Ser271 in differentiated MM6 cells, interestingly regardless of cell stimulation.
Finally, the primary amine derivatization procedure by EZ-linkTM was utilized for MS analysis of lysine rich proteins. In the past, chemical propionylation of histones had been employed prior to trypsin digestion; however it was easily confused in MS with combinations of other PTMs (e.g. acetylation, methylation). Moreover, propionylation is a PTM for histone H3 and this information was lost. Consequently, the EZ-link reagent was more useful for analysis of histones, as unambiguous assignment of PTMs and detection of native propionylation on bovine H3 became possible.
Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.
Als am 11.10.1987 der Stern- Reporter Sebastian Knauer den toten, erst kurz zuvor vom Amt des Ministerpräsidenten in Kiel zurückgetretenen, Uwe Barschel (CDU) in der Badewanne seines Genfer Hotelzimmers fotografierte1, schuf er eine Fotografie, deren Bekanntheit und Streitbarkeit bis heute nicht verblasst ist.