Death-associated protein kinases

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Investigations on the role of mitogen-activated protein kinases 11 and 17 in defense response of Arabidopsis thaliana

Investigations on the role of mitogen-activated protein kinases 11 and 17 in defense response of Arabidopsis thaliana

Effector proteins are usually recognized by intracellular receptor proteins belonging to the nucleotide binding-leucine-rich repeat (NB-LRR) family [Luck et al., 2000; Caplan et al., 2008]. The activation of ETI leads to programmed cell death, the so-called hypersensitive response (HR), and often to systemic acquired resistance (SAR) (see below). In order to evade host de- fence mechanism, pathogens evolve with disguised or altered effector molecules, putting the selection pressure again on the plants. This coevolution between plants and plant pathogens is summarized in the so called “zigzag model of plant immunity” by Jones and Dangl [2006]. Recent research demonstrates that the zigzag model might oversimplify the situation in real life [Boller and Felix, 2009; Thomma et al., 2011]. For example, there are several PAMPs that induce HR [Wei et al., 1992; Naito et al., 2007]. In addition to PAMPs and effector molecules, so called “damage-associated molecular patterns” (DAMPs) play an important role in plant- pathogen interactions [Boller and Felix, 2009]. These molecules are products generated by pathogen enzyme activity and function as endogenous elicitors. Examples for DAMPs are plant cell wall fragments [Darvill et al., 1994], cutin monomers [Kauss et al., 1999], and the peptides systemin in tomato [Pearce, 2011] and AtPep1 in Arabidopsis [Huffaker et al., 2006].
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On the role of superoxide dismutase and protein phosphatase type-2C beta in neuronal cell death

On the role of superoxide dismutase and protein phosphatase type-2C beta in neuronal cell death

About 600 kinases are known to be encoded by the human genome but only an estimated number of 200 phosphatases (Cohen, 2001). However, since single phosphatase catalytic units are often associated with several different regulatory or targeting subunits, it is believed that the number of functional phosphatase holoenzymes adds up to the number of protein kinases. Whereas, so far, the focus of research interest was predominantly on protein kinases, it is evident that protein phosphatases as their counterparts play an equally important role. There are 4 potential sites for phosphorylation and dephosphorylation in proteins: the amino acids serine, threonin and tyrosine which contain a free –OH residue whose phosphorylation leads to O-phosphates and histidine which can be phosphorylated at its -NH group creating N- phosphoamidates. Thus, 3 different superfamilies of protein O-phosphatases exist: The protein tyrosine phosphatases (PTP) including members such as dual-specifity phosphatase and serine/threonine phosphatases which are grouped into members of the PPP family (phosphoprotein phosphatases) and PPM family (protein phosphatase Mg 2+ -dependent) which are defined by molecular structure and conserved amino acid sequences (Klumpp et al., 2002). PP1, PP2A or PP2B belong to the family of PPPs whereas five PP2C isoforms and the pyruvate dehydrogenase with similar amino acid sequences are part of the PPM family (Ingebritsen and Cohen, 1983). Additionally, protein histidine phosphatases have been discovered which can dephosphorylate N-phospho-histidine. The nomenclature of serine/threonin phosphatases has been established according to their biochemical properties before gaining more knowledge about structural similarities: Type 1 enzymes were inhibited by heat-stable inhibitor proteins and preferentially dephosphorylated the ß-subunit of phosphorylase kinase. Type 2 protein phosphatases, on the contrary, were insensitive to these inhibitors and preferentially dephosphorylated the alpha unit of the phosphorylase kinase. Type 2 was divided in different subtypes which were activated under special conditions: While PP2A was spontaneously active, PP2B activity was Ca 2+ -dependent and PP2C Mg 2+ -
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The death-associated protein kinase 1 (DAPK1) : prognostic relevance in pediatric acute lymphoblastic leukemia (ALL) and evaluation as a therapeutic target

The death-associated protein kinase 1 (DAPK1) : prognostic relevance in pediatric acute lymphoblastic leukemia (ALL) and evaluation as a therapeutic target

DAPK1 is the most prominent member belonging to a family of Serine/Threonine (Ser/Thr)-kinases, involved in cell death associated mechanisms (Bialik, Kimchi 2006; Deiss et al. 1995). With a size of about 160 kDa, DAPK1 is a multi-domain protein, consisting out of several regions playing different roles in regulation processes and execution of its pro-apoptotic and anti-metastatic functions (Bialik, Kimchi 2006; Cohen et al. 1997; Inbal et al. 1997; Singh et al. 2016). In more detail, DAPK1 has a conserved catalytic/kinase-domain, which is regulated by an autoregulatory Ca 2+ /Calmodulin-dependent-domain and accounts for phosphorylation of different substrates like Myosin-Light-Chain (MLC) (membrane blebbing via actin activation), Syntaxin-1 A (vesicle/membrane fusion) or even promotes autophosphorylation (Cohen et al. 1997; Bialik, Kimchi 2004; Bialik, Kimchi 2006; Shohat et al. 2001). Furthermore, adherence to actin filaments of the cytoskeleton is provided by the “cytoskeletal binding region” and a region containing several Ankyrin-repeats is involved in proper DAPK1 localization and its degradation (Bialik et al. 2004; Bialik, Kimchi 2006; Nair et al. 2013; Singh et al. 2016). Importantly, DAPK1 exhibits a death-domain, that is described to promote protein-protein interactions, as well as influence of kinase activity and apoptotic functions (Singh et al. 2016). Furthermore, bidirectional interactions with ERK (Extracellular-signal-Regulated-Kinase) are shown to be associated with apoptotic effects of DAPK1 (Chen et al. 2005). Moreover, DAPK1 was also described to play a role in cell death induction via autophagy while it is involved in formation of autophagic vesicles (Inbal et al. 2002; Levin-Salomon et al. 2014; Singh et al. 2016; Zalckvar et al. 2009).
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Characterization of the Proline-rich Synapse-associated Protein 1 Knockout Mouse

Characterization of the Proline-rich Synapse-associated Protein 1 Knockout Mouse

In order to understand the PSD (ultra)structure and the dynamic changes in the PSD subcompartment, the identification and characterization of individual PSD proteins is of enormous importance. From numerous studies it is believed that the PSD is composed of a huge complex protein network consisting of several hundred different proteins (Collins et al. 2006; Cheng et al. 2006). The knowledge of these proteins was ameliorated in the year 2000 by Walikonis and coworkers as they combined SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separation of PSD proteins with MALDI-TOF mass spectrometry and identified and partially sequenced about 30 proteins that are significantly enriched in PSD preparations. The resulting complex protein assembly is the molecular basis for various intercellular occurrences. These events include the clustering and transport of membrane bound receptors, the organisation of signalling cascades, the dynamic organization of cytoskeletal components and the induction and structural maintenance of intercellular contact sites.
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Modulation of the Unfolded Protein Response by Kaposi's Sarcoma-associated herpesvirus

Modulation of the Unfolded Protein Response by Kaposi's Sarcoma-associated herpesvirus

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is a member of the large Herpesviridae family. As with the other family members, two stages to its life cycle have been detected, namely latency and lytic replication. The main state of KSHV is latent infection with only a small population undergoing lytic replication. Most of the viral proteins are expressed during lytic infection which results in the production of progeny virions. Like other viruses, KSHV modulates several cellular signaling pathways for its own benefit. It is known that endoplasmic reticulum (ER) stress and consequently activation of the unfolded protein response (UPR) triggers KSHV lytic infection, however little is published about how KSHV modulates the UPR. Upon accumulation of unfolded and misfolded proteins in the ER (as it can occur during viral infection), cells trigger a signaling pathway in order to restore ER homeostasis, designated as UPR. The UPR consists of three ER-to-nucleus signaling pathways that regulate synthesis, folding, and degradation of proteins in the ER. One of the three UPR sensors, IRE1, activates the transcription factor XBP1, which induces the expression of chaperones and ER-associated degradation factors, thereby alleviating ER stress. Although ER stress can trigger lytic KSHV replication by reactivating the KSHV replication transcription activator factor (RTA) promoter via XBP1s, the effect of KSHV on the UPR is not clearly understood. The aim of this study was to investigate the influence of a lytically replicating KSHV (KSVH LYT ) on the UPR via the IRE1 signaling pathway. Here I
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Mechanoactivation of protein kinases

Mechanoactivation of protein kinases

PER. After addition of Lysozyme and DNase cells were fully lyzed by sonification. Insoluble cell debris as well as inclusion bodies were sedimented by centrifugation at 20000 g for 30 min. After discarding the supernatant the inclusion body containing pellet was again resuspended in 4 ml / 1 g washing buffer (30 mM Tris HCl pH 7.5, 150 mM NaCl and 0.1% TritonX-100). Centrifugation and washing of the inclusion bodies were repeated four times, when the supernatant appeared fairly cleared. The inclusion bodies containing the Cys-modified functional ST were then dissolved in 6 ml solubilization buffer (20 mM Tris HCl pH 7.5, 6 M Guanidinium HCl), the ones containing the non- functional and untagged variant in 12 ml. After determining the protein concentration in the solubilized fractions by measuring the absorbance at 280 nm, the entire amount of non-functional ST was used and mixed with the volume equivalent of a tenth in mass of the latter with functional 6xHis-mono-Cys-ST. The mixed solubilized protein was again subjected to centrifugation for 30 min at 20000 g and the supernatant with the unfolded ST constructs collected. To accomplish refolding the mixture was slowly and drop-wise added to a stirred reservoir of 500 ml 1x PBS and 10 mM β-Mercaptoethanol (the use of DTT or the more expensive TCEP as reducing agents is also possible, if compatible with the Ni2+-column matrix used for the following His-Tag affinity purification step). The mixture was stirred over night at 4 °C to maximize refolding of the mixed ST. Next, the 500 ml protein sample was filtered through a cellulose filter to remove precipitate and then loaded onto a 5 ml HisTrap FF column (GE Healthcare) for Ni-IMAC purification. Elution of the reassembled monoST was achieved by a linear gradient from 10 to 300 mM Imidazole (in 1x PBS, 10 mM β-Mercaptoethanol). Elution fractions were analysed in gel electrophoresis. If the samples were not heated in gel loading dye prior to loading
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The amyloid precursor protein potentiates CHOP induction and cell death in response to ER Ca2+ depletion

The amyloid precursor protein potentiates CHOP induction and cell death in response to ER Ca2+ depletion

Here we investigated the role of the amyloid precursor protein (APP) in regulation of Ca2+ store depletion- induced neural cell death. Ca2+ store depletion from the endoplasmic reticulum (ER) was induced by the SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) inhibi- tor thapsigargin which led to a rapid induction of the unfolded protein response (UPR) and a delayed activa- tion of executioner caspases in the cultures. Overexpres- sion of APP potently enhanced cytosolic Ca2+ levels and cell death after ER Ca2+ store depletion in comparison to vector-transfected controls. GeneChipR and RT-PCR anal- ysis revealed that the expression of classical UPR chaper- one genes was not altered by overexpression of APP.Interestingly, the induction of the ER stress-respon- sive pro-apoptotic transcription factor CHOP was signifi- cantly upregulated in APP-overexpressing cells in comparison to vectortransfected controls. Chelation of intracellular Ca2+ with BAPTA-AM revealed that enhanced CHOP expression after store depletion occured in a Ca2+-dependent manner in APPoverexpressing cells. Prevention of CHOP induction by BAPTA-AM and by RNA interference was also able to abrogate the potentiat- ing effect of APP on thapsigargin-induced apoptosis. Application of the store-operated channel (SOC)-inhibi- tors SK F96365 and 2-APB downmodulated APP-triggered potentiation of cytosolic Ca2+ levels and apoptosis after treatment with thapsigargin. Our data demonstrate that APP-mediated regulation of ER Ca2+ homeostasis signifi- cantly modulates Ca2+ store depletion-induced cell death in a SOC- and CHOP-dependent manner, but independ- ent of the UPR.
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Death interrupted: Contemporary economies of death and dying

Death interrupted: Contemporary economies of death and dying

tality itself,” as Timmermans observed, “cannot be avoid- ed, but individual causes of death can be determined, and then manipulated and postponed” (2006: 11). Thus, when a first line of chemotherapy fails, oncologists can offer second, third, and even fourth lines. When patients’ kid- neys fail, nephrologists can treat them with dialysis for years, and surgeons can offer them transplants. When patients stop breathing, physicians can connect them to a respirator. And when the heart stops, doctors can attempt resuscitation. Dying, in other words, often involves deci- sion-making: for a person to die, active decisions are made not to conduct these procedures (Agamben, 1998; Calla- han, 1987; Zussman, 1993). This plethora of interventions is extremely expensive, and given the pricelessness of life, it raises the specter of unsustainable spending (Ubel, 2000). The field of Health Economics has gained much influence in policymaking in tandem with the increased availability of these medical interventions. In the United Kingdom, the Quality-Adjusted Life Years (QALY) metric has standardized the benefit of medical treatment and rationalized resource allocation (Ashmore et al. 1989); today, with several excep- tions, the UK’s National Health Service agrees to pay 20,000–30,000 British pounds for one standardized life year. This rational care-rationing mechanism has been criticized from various standpoints, but the imperative to ration care in some way or another remains, and healthcare systems necessarily address it, implicitly if not explicitly. How much to spend on prolonging the lives of severely ill patients has become an economic as well as a moral question, even in the U.S. healthcare economy, where no central mechanism for care rationing is applied (Callahan, 1987; Livne, 2014).
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Inhibition of bone morphogenetic protein signal transduction prevents the medial vascular calcification associated with matrix gla protein deficiency

Inhibition of bone morphogenetic protein signal transduction prevents the medial vascular calcification associated with matrix gla protein deficiency

exogenous BMP-2. (B) Cultured aortic VSMCs from wild-type mice were transfected with either scrambled siRNA (siSC) or siRNA targeting MGP (siMGP) at 20 nM. RNA was isolated from cells after 4 days. siMGP decreased MGP mRNA levels in WT VSMCs by >95% compared with siSC-treated cells. However, depletion of MGP in WT VSMCs did not alter Id1 mRNA levels. **P<0.0001 compared to siSC-treated VSMCs. (C) VSMCs isolated from wild-type mice were treated with 20 nM of either scrambled siRNA (siSC) or siRNA specific for MGP (siMGP). Cells were incubated with or without BMP-2 (20 ng/mL) for 1 h prior to protein harvest. Western blots were probed with antibodies specific for phosphorylated Smad 1/5 (P-Smad 1/5) and total Smad 1. Depletion of MGP in WT VSMCs did not alter the ratio of P-Smad 1/5 levels to total Smad 1 levels, both at baseline and in response to exogenous BMP-2.
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Functional characterization of the mitotic-spindle and kinetochore associated protein astrin

Functional characterization of the mitotic-spindle and kinetochore associated protein astrin

To ensure that each newly formed daughter cell receives a complete genome progression through the cell cycle is controlled by a series of biochemical switches that trigger the events of the cell cycle in the correct sequence. The central components of the cell cycle control system are the cyclin-dependent protein kinases (Cdks), which are controlled by transient associations with cyclin regulatory subunits, phosphorylation and inhibitory proteins. Concentrations of Cdks are constant throughout the cell cycle and their activities depend primarily on the changing levels of the associated cyclins. Based on the timing of expression and their function, the cyclins can be divided into four classes, G1 cyclins, G1/S cyclins, S cyclins and M cyclins. G1 cyclins (D-type cyclins in vertebrates) bind and activate Cdk4 and Cdk6, which stimulate the entry into a new cell cycle in response to external factors. G1/S cyclins (cyclin E in vertebrates) activate Cdk2 and trigger G1/S transition; their concentrations peak in late G1. S cyclins (cyclin A in vertebrates) bind Cdk2 and Cdk1 and are necessary for DNA synthesis; their concentrations rise and remain high during S phase, G2 and early mitosis. Cdk1 in association with the M cyclin, cyclin B1, is the key regulator of both mitotic entry and progression through mitosis (Murray, 2004).
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Effect of tyrosine autophosphorylation on catalytic activity and subcellular localisation of homeodomain-interacting protein kinases (HIPK)

Effect of tyrosine autophosphorylation on catalytic activity and subcellular localisation of homeodomain-interacting protein kinases (HIPK)

Erklärung über den Eigenanteil der Arbeit Hiermit erkläre ich, Jan van der Laden, an Eides statt, dass ich den wesentlichen Anteil an der Publikation: van der Laden J, Soppa U, Becker W: Effect of tyrosine autophosphorylation on catalytic activity and subcellular localisation of homeodomain-interacting protein kinases (HIPK); Cell

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The effect of translationally controlled tumour protein (TCTP) on programmed cell death in plants

The effect of translationally controlled tumour protein (TCTP) on programmed cell death in plants

The effect of AtTCTP on the pro-apoptotic effect of BAX, a member of the Bcl-2 family of mouse, was tested in tobacco leaves (Figure 1). Transient expression in N. benthamiana was achieved through Agrobacterium tumefaciens and relative ion leakage was measured for cell death quantification. Expression of cytotoxic mouse BAX was induced by floating transformed tobacco leaf discs on 2 μM dexamethasone. AtTCTP was constitutively expressed by the CaMV 35S promoter. 28 hours after induction of BAX expression an increase in ion leakage was observed, that progressively increased until the end of experiment at 69 hours post induction. Co-expression of AtTCTP strongly reduced BAX mediated cell death. Statistical analyses were carried out using analysis of variance (P > 0.05) and are shown in Figure 1A. Expression of AtTCTP over time was observed using Western blot analyses. As shown in Figure 1B, AtTCTP amounts remain stable when expressed without BAX, while BAX co-expression resulted in a slow decrease of AtTCTP over time. As shown in the left panel of Figure 1C, BAX expressing leaf discs revealed clear signs of chlorosis after 69 hours of incubation on 2 μM DEX, whereas AtTCTP over-expressing ones did not. Co-expression of BAX and AtTCTP resulted in slightly chlorotic discs. Trypan blue staining, used to visualize dead cells within leaf discs, provided comparable results (panels on the right side of Figure 1C). The highest amount of dead cells was found in BAX over-expressing tissues, followed by BAX and AtTCTP co-expressing tissues, and AtTCTP over-express- ing leaf disc showed almost no dead cells. Control experi- ments using leaf discs floating on 2 μM dexamethason and expressing either non-apoptotic ΔC-BAX (see [25]), or StrepII-tag without fusion protein, or a combination of ΔC-BAX and AtTCTP are shown in Figure 1D. In contrast to full length BAX, the truncated form (ΔC-BAX) lacks the C-terminal transmembrane region needed for mitochondrial targeting. Therefore, ΔC-BAX is not able to cause cell death (compare [25]). No statistically sig- nificant differences were found between controls (analysis of variance).
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Functional characterization of B-cell receptor associated protein 31 in cancer cells

Functional characterization of B-cell receptor associated protein 31 in cancer cells

As NFκB is connected to the autophagy process in cancer cells (Zhu et al., 2017), we investigated if also in our model we can observe the difference between the wild type and BAP31 knockout cells. It is known, that autophagy is a very dynamic process, therefore it is not possible to just check the expression of the proteins associated with autophagy. Zhang et al. (2018) provide an instruction how to properly evaluate the autophagic flux. Rephrasing this article, state of the art method requires to use the inhibitor of autophagy (we used Concanamycin) next to the control cells. Subsequently, it is recommended to subtract the amount of expressed control protein from the one treated with the inhibitor. Then, the result of the equation gives the amount of autophagic flux.
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Yes-associated protein (YAP) expression and its biological role in thyroid
gland

Yes-associated protein (YAP) expression and its biological role in thyroid gland

Blocking prevents the background staining resulting from any non-specific binding of the anti- body to endogenous peroxidase, alkaline phosphatase, avidin and biotin. Epitope recovery step is often required for formalin fixed paraffin-embedded samples to unmask the epitopes that are altered during fixation and processing. For this purpose, a number of heat-based treatment tech- niques have been developed including water baths, microwaves, autoclaves, and pressure cook- ers. The heat can unmask epitopes by disrupting cross-links formed by formalin fixation and by changing the tertiary protein structure. Among these heating methods, water bath system has shown fairly good results for a wide range of antibodies and tissues. For certain antibodies in which heat is not recommended, enzymatic treatment using trypsin or pepsin may be an alterna- tive. Next, the target antigen is detected by direct enzyme-based techniques such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The antigen-antibody complex is finally visual- ized by the addition of a chromogen such as 3,3`-diaminobenzidine (DAB) or 3- amino-9- ethylcarbazole (AEC), which stains brown or red respectively. The choice of chromagen depends on colour preference and solubility. Where not specified, steps are performed at room tempera- ture. Positive and negative controls should be used in each assay to ensure quality staining (Ha- yat 2006).
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Assoziation von SNARE-associated Protein Snapin (SNAPAP) mit
Negativsymptomatik und kognitiven Defiziten bei schizophrenen Patienten

Assoziation von SNARE-associated Protein Snapin (SNAPAP) mit Negativsymptomatik und kognitiven Defiziten bei schizophrenen Patienten

„Ich, Martin Liening, versichere an Eides statt durch meine eigenhändige Unterschrift, dass ich die vorgelegte Dissertation mit dem Thema „Assoziation von SNARE- associated Protein Snapin (SNAPAP) mit Negativsymptomatik und kognitiven Defiziten bei schizophrenen Patienten“ selbstständig und ohne nicht offengelegte Hilfe Dritter verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel genutzt habe. Alle Stellen, die wörtlich oder dem Sinne nach auf Publikationen oder Vorträgen anderer Autoren beruhen, sind als solche in korrekter Zitierung (siehe „Uniform Requirements for Manuscripts (URM)“ des ICMJE –www.icmje.org) kenntlich gemacht. Die Abschnitte zu Methodik (insbesondere praktische Arbeiten, Laborbestimmungen, statistische Aufarbeitung) und Resultaten (insbesondere Abbildungen, Graphiken und Tabellen) entsprechen den URM (s.o) und werden von mir verantwortet.
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Structural Characterization of Small basic protein (Sbp) and Accumulation associated protein (Aap) : two Proteins involved in Biofilm Formation in Staphylococcus epidermidis

Structural Characterization of Small basic protein (Sbp) and Accumulation associated protein (Aap) : two Proteins involved in Biofilm Formation in Staphylococcus epidermidis

In vitro folding of a protein may not necessarily reflect its folding state in vivo; however, it was necessary to see the secondary structure and folding state of Sbp before crystallization experiments. Different homology models of Sbp were created by bioinformatics tools. Although each model designed by a particular tool was different due to very low sequence homology with proteins of already known structures, but all of them suggested the presence of a high percentage of β-strands, some parts of α-helices and a large part consisting of coils. The stability of Sbp in solution, in terms of folding state, was examined by CD spectrometry, which revealed an overall partially folded protein with a composition of β-sheets (45 %), helices (2 %) and random coil regions (51 %), according to Yang’s algorithm (Yang et al., 2015). Reed’s algorithm (Greenfield, 2006) showed an approximation of 46 % β-sheets, 6 % α-helices and 33 % random coil. Both these references are not only in good agreement with each other but also support the credibility of the secondary structure prediction by bioinformatics tools to some extent. The aggregation behavior of Sbp can be correlated to the fact that β-sheet rich proteins mostly involve more intermolecular interactions resulting in aggregation of proteins (Fink, 1998)
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Death in Modern Germany

Death in Modern Germany

Kerstin Rehwinkel explored the origins of a new attitude toward death during and after the Enlightenment in her paper on “‘Apparent Death’ Discourse in Germany in the Eighteenth and Nineteenth Century: Body, Science, and Society.” Rehwinkel noted that the discourse about “apparent death” began in Paris in the 1740s, leading to the creation of life-saving institutions and methods of treating the “apparently” dead all over Europe, with Hamburg being the first German city to implement them. Reformers only gradually succeeded in overcoming the general public’s fear of becoming contaminated by touching the dead, however, particularly the corpses of suicides. Rehwinkel pointed out that as phy- sicians replaced priests as the final authority on death, the question of how exactly to define dying and death took on a new meaning. Simone Ameskamp examined the rise of cremation in her paper “Phoenix and Prometheus—Cremation in Imperial and Weimar Germany.” The first crematorium in modern Germany was built in Gotha in 1878. Urban Protestants constituted the backbone of the cremation movement. They emphasized the modernity, cleanliness, and dignity of the practice, and linked it to German values. While their Protestant opponents rejected cremation mainly as not in line with tradition, Catholics until 1963 were supposed to heed a papal decree banning it. Drawing upon statistical evidence that showed a steady growth of cremation, Ameskamp argued that the world wars had little impact on its acceptance.
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On the influence of the protein kinases pCKII, STN7 and STN8 on chloroplast phosphorylation networks in Arabidopsis thaliana : [kumulative Dissertation]

On the influence of the protein kinases pCKII, STN7 and STN8 on chloroplast phosphorylation networks in Arabidopsis thaliana : [kumulative Dissertation]

spectrometry approaches are developed for this purpose, whereof most of them are nowadays performing in high resolution with the ability of detecting ions in femtomolar range, the improved mass accuracy till a few parts per million (ppm) for the peptide and the acceleration of duty cycles (Reiland et al., 2011). Phosphopeptides are in general hard to detect via mass spectrometry due their negative charge, which impedes their detection in the positive ion modes, and their hydrophilic nature, which hinders proper binding to the columns (Mann et al., 2002). Most phosphorylation events occur at low abundance, which means that only a part of the protein entity is affected. This results in peaks of low intensity, especially when the non-phosphorylated peptide is present leading to ionic suppression (Mann et al., 2002). This low stoichiometry necessitates enrichment strategies of phosphopeptides as prerequisites for sufficient MS detections (Linding et al., 2007). Roughly a half of the plant phosphoproteomics studies used titanium dioxide (TiO 2 ) for phosphopeptide enrichment and the other half a diversity of immobilized metal affinity chromatography (IMAC) strategies (van Wijk et al., 2014). Reliable phosphorylation studies via MS need a robust sample preparation procedure and high accuracy MS detections. In liquid chromatography tandem MS (LC-MS/MS) approaches is the peptide identification capacity limited by the chromatographic performance of the LC-system, which is determined by its peak capacity and separation efficiency. Furthermore are the MS-scan rate and sensitivity important parameters for reliable fragment detections over a wide dynamic range (Macek et al., 2009).
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Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and the Bcl-2 Protein Family

Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and the Bcl-2 Protein Family

expressed receptor on epithelial cells but has also been found on monocytes [30, 31]. One well-known EGFR ligand is amphiregulin (AREG) [32]. AREG is a growth factor of the EGF family [33], which was shown to be expressed on monocytes [34] and T cells [35] and to control the inflammation process [36]. Synthesized as the transmembrane precursor pro-AREG, the soluble protein is released by limited proteolytic cleavage of the precursor [37]. In a previous study, we demonstrated that CBMO have a higher pro-AREG surface expression compared to PBMO. Hence, upon E. coli infection-induced cleavage of pro-AREG, CBMO show an 11-fold higher level of soluble AREG compared to PBMO [38]. We further showed that AREG increases intracellular MMP-2 and MMP-9 levels and induces cleavage of membrane-bound FasL through engagement with the EGF receptor, pointing towards involvement of the extrinsic apoptosis pathway. Reduction of AREG levels was found to diminish PICD in CBMO and PBMO [38].
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Effects of Adenosine-3': 5 '-monophosphate (cAMP) on the Activity of Soluble Protein Kinases in Maize {Zea mays) Coleoptile Homogenates 

Effects of Adenosine-3': 5 '-monophosphate (cAMP) on the Activity of Soluble Protein Kinases in Maize {Zea mays) Coleoptile Homogenates 

B. Janistyn • E ffects of A d en osin e-3':5'-m on op h osp h ate (cA M P ) 583 protein extracts in the assay. The corrected values are shown in Fig. 3 (see text). T here are seven differ­ ent protein kinases, P I to P V II visible. P I, P II and P VI are activated by 6 |im cAM P. P H I, P IV and P V seem to be cAM P independent while P V II is in­ creased in the absence of cAM P. The diagram of the protein kinase assays are in good agreem ent with the diagrams shown in Figs. 1 and 2. But evidently the activity of the enzymes has been decreased through the extraction procedure. In particular the cAM P de­ pendent protein kinases are mostly decreased in their activity. Therefore it is not astonishing that so far no cAM P dependent protein kinase could be isolated from higher plants in it’s substantial form. Recently, S. Ejiri et al. [20] reported effects of cAM P and cGM P on the autophosphorylation of elongation fac­ tor 1 from wheat embryos. A t 10-7 m cGM P stim u­ lated and cAM P inhibited the phosphorylation.
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