Functional analysis of strong MAMP-responsive synthetic promotors

Microbe-associated molecular pattern (MAMP) sind zum Beispiel Moleküle von Pathogenen, die bei Pflanzen eine durch cis-regulatorische Sequenzen vermittelte Hochregulation von Abwehrgenen bewirken. Grundlage der vorliegenden Arbeit war die vorangegangene Identifizierung neuer MAMP-responsiver cis-Sequenzen aus Arabidopsis thaliana. Das Ziel der vorliegenden Arbeit ist die funktionelle Analyse der zwei stärksten MAMP-responsiven cis-Elemente. Dabei handelt es sich zum einen um das cis-regulatorische Modul (CRM) des DJ1E-Gens und zum anderen um die MAMP-responsive Sequenz (MRS) des At1g13990-Gens. Das CRM-DJ1E ist essentiell für die MAMP-responsive Genexpression des Gens DJ1E. Es enthält drei funktionelle MAMP-responsive cis-Sequenzen, zwei WT-Boxen GGACTTTT und GGACTTTG sowie eine GCC-ähnliche Box GCCACC. Das CRM-DJ1E ist Salizylsäure (SA)-responsiv, wobei die GGACTTTT-Box essentiell ist. Die GGACTTTG und GCCACC-Box sind notwendig für die volle SA-Induktion. Die in dieser Arbeit identifizierten AP2/ERF-Transkriptionsfaktoren (TF) ORA59 und ERF10 interagieren antagonistisch mit der GCC-ähnlichen Box des CRM-DJ1E, wobei es sich bei ORA59 um einen Aktivator und bei ERF10 um einen Repressor handelt. Die Kernsequenz GCCNCC ist für die Interaktion mit ORA59 essentiell. TF die mit den WT-Boxen interagieren konnten nicht identifiziert werden. Die MRS-At1g13990 liegt in einer außergewöhnlich kurzen intergenischen Region. Sie ist für eine MAMP-vermittelte Genexpression von At1g13990 ausreichend, wobei die 3´UTR des upstream liegenden Gens noch zusätzlich einen quantitativen Effekt aufweist. Die MRS-At1g13990 enthält eine 10 bp lange, für die MAMP-Responsivität essentielle Kernsequenz TCGTTTACGT. Für eine voll ausgeprägte MAMP-vermittelte Genexpression ist jedoch die gesamte MRS nötig. In transgenen A. thaliana-Pflanzen vermittelt das CRM-DJ1E sowie die MRS-At1g13990 Pathogen-induzierte Reportergenaktivität gegenüber einem virulenten und avirulenten Pseudomonas syringae Stamm und dem nekrotrophen Pilz Botrytis cinerea. Die beiden Pseudomonas Stämme zählen zu den biotrophen bzw. hemi-biotrophen Pathogenen. Die erhöhte Basalaktivität der MRS-At1g13990 demonstriert die niedrige Spezifität des synthetischen Promotors. Im Vergleich dazu ist die Pathogen-induzierte Reportergenexpression der transgenen CRM-DJ1E-Pflanzen präzise um die Infektionsstelle lokalisiert. Es ist kaum eine Hintergrundexpression zu erkennen. Somit ist dieses Element vermutlich geeignet für die Entwicklung Pathogen-resistenter Pflanzen, bei denen es zu einer Induktion von Zelltod-vermittelnder Gene kommt. Für das bessere Verständnis der Wirkungsweise dieses Elementes ist eine Identifikation der übrigen WT-Box-bindenden TF von Vorteil.Microbe-associated molecular pattern (MAMP) are for example molecules from pathogens that upregulate defense genes through specific cis-regulatory sequences. The basis of this work was the previous identification of novel MAMP-responsive cis-sequences from Arabidopsis thaliana. The aim of this work is the functional dissection of the two strongest MAMP-responsive cis-elements, the cis-regulatory module (CRM) of the DJ1E gene and the MAMP-responsive sequence (MRS) of the At1g13990 gene. The CRM-DJ1E is essential for the MAMP-responsive gene expression of the DJ1E gene. It has three functional MAMP-responsive cis-sequences, two WT-boxes GGACTTTT and GGACTTTG and a GCC-like box GCCACC. The CRM-DJ1E is salicylic acid (SA)-responsive, whereby the GGACTTTT-box is essential and the GGACTTTG and GCCACC-box are necessary for the full SA-induction. In this work, the transcription factors (TF) ORA59 and ERF10 were identified to interact antagonistically with the CRM-DJ1E. ORA59 is an activator and ERF10 a repressor of the CRM-DJ1E. The core sequence GCCNCC is essential for the interaction with ORA59. TF interacting with the WT-boxes were not identified. The MRS-At1g13990 is located in an extraordinarily short intergenic region. It is sufficient for the MAMP-responsive gene expression of At1g13990. Furthermore the 3´UTR-Region of the upstream gene has a quantitative effect on the MAMP-induced gene expression. The MRS-At1g13990 contains an essential core-sequence TCGTTTACGT, which is 10 bp long. For a full MAMP-responsive gene expression the whole MRS is necessary. Transgenic A. thaliana plants harbouring the CRM-DJ1E and MRS-At1g13390 show pathogen-induced reporter gene activity in response to virulent and avirulent Pseudomonas syringae strains and to the necrotrophic fungus Botrytis cinerea. Both Pseudomonas strains are hemi-biotrophic. The high background activity of the MRS-At1g13990 under non-infected conditions demonstrates a low specificity of the synthetic promoter. In contrast, the CRM-DJ1E shows pathogen-induced reporter gene expression precisely located around the infected area. Therefore, it may be suitable for the development of pathogen resistant plants by inducing cell death mediating genes. To better understand the function of this element an identification of the WT box-binding transcription factors is of advantage

Ein zelluläres Entzündungsmodell zur Untersuchung Chlorid-abhängiger Sensibilisierungsprozesse in Schmerzzellen

Die Kenntnis über die Mechanismen der Schmerzverarbeitung und ihre Veränderung durch Entzündungen spielt eine wichtige Rolle in der Entwicklung spezifischer Schmerzmedikamente. Schmerzreize werden von den Fasern spezialisierter Nervenzellen des peripheren Nervensystems, den Nozizeptoren detektiert, deren Zellkörper in den Spinalganglien liegen. Die Nozizeptoren selbst stellen somit die erste Verarbeitungsstelle für Schmerzreize dar. Die Erforschung der Beeinflussung ihrer Signaltransduktion durch Entzündungsmediatoren stand deshalb im Mittelpunkt dieser Arbeit. Hier ist es gelungen ein neues Modell auf zellulärer Basis zu entwickeln, das die Induzierung einer Entzündung innerhalb weniger Stunden ermöglicht und sowohl ex vivo als auch in vitro angewendet werden kann. Mit Hilfe des Modells ist es gelungen den Einfluss einer Entzündung auf die Aktivität und Expression verschiedener Transduktionskanäle zu untersuchen. Ebenso wurde die Rolle von Chlorid in der Signaltransduktion von Schmerzzellen analysiert. Es konnte gezeigt werden, dass Entzündngsmediatoren zu einer vertärkten Signaltransduktion und damit möglicherweise auch zu einer gesteigerten Schmerzwahrnehmung führen

Ethics education in nursing—structural characteristics and didactical implications of nursing education

Die Pflegeausbildung weist die Besonderheit auf, dass die berufliche Bildung an unterschiedlichen Lernorten erfolgt. Die jeweils beteiligten Lernorte (Lernort Theorie, Lernort Praxis, zunehmend erweitert durch den dritten Lernort, das Skillslab) beeinflussen und fördern die Entwicklung der Ethikkompetenzen der angehenden Pflegfachpersonen – lernortspezifisch als auch lernortübergreifend – in unterschiedlicher Weise. Diese besonderen strukturellen Gegebenheiten des Lehrens und Lernens wirken sich sowohl auf die Ausgestaltung der Ethikbildung als auch auf die Förderung der Ethikkompetenzentwicklung im Ausbildungsverlauf aus. Die Ausführungen leitet die folgende Frage: Welche spezifischen pädagogischen und didaktischen Anforderungen, aber auch welche bildungsrelevanten Rahmungen ergeben sich angesichts der unterschiedlichen Lernorte für die Ethikbildung und die Ethikkompetenzentwicklung zukünftiger Pflegefachpersonen? Der Beitrag unterstreicht die Relevanz einer systematischen, methodisch reflektierten und lernortabgestimmten Ethikbildung im Bereich der Pflegeausbildung wie auch die Relevanz der bewussten Einbindung ethischer Reflexionsräume in die Prozesse der Ethikkompetenzentwicklung an den jeweiligen Lernorten. Die Besonderheiten der Ethikbildung erschließen sich hierbei aus den pflegeberuflichen Anforderungen an das professionelle Pflegehandeln und aus den Erfahrungen der Lernenden an den unterschiedlichen Lernorten im Verlauf der Pflegeausbildung.Background: A special characteristic of nursing education is that professional teaching takes place in various locations. The learning locations involved (such as learning in theory, learning in clinical practice, or learning in third facilities, which are becoming increasingly popular, such as the skills lab) have an impact on and promote the development of ethical competences among prospective nursing professionals in various ways. Furthermore, it has become evident that the structural conditions that are specific to teaching and learning in nursing education programs shape the organization of ethics education and the promotion of ethical competence development among nurse trainees over the course of their education. Objectives: The aim of this article is to discuss the following key question: What are the specific pedagogical and didactical requirements and educational frameworks that arise in view of the various learning locations in nursing education in order to facilitate ethics education and ethical competence development among prospective nursing professionals? Discussion: This article emphasises the importance of a systematic, methodically reflected and learning-location-coordinated approach to ethics education in the field of nursing education. Moreover, it highlights the relevance of the conscious promotion of settings for ethical reflection within the process of ethical competence development in the different locations of learning. The unique quality of ethics education in nursing emerges from the professional requirements that are characteristic to nursing care and the individual experiences of trainees at the various locations of learning throughout their nursing education progra

Acupuncture in acute herpes zoster pain therapy (ACUZoster) - design and protocol of a randomised controlled trial

Background: Acute herpes zoster is a prevalent condition. One of its major symptoms is pain, which can highly influence patient's quality of life. Pain therapy is limited. Acupuncture is supposed to soften neuropathic pain conditions and might therefore act as a therapeutic alternative. Objective of the present study is to investigate whether a 4 week semi-standardised acupuncture is non-inferior to sham laser acupuncture and the anticonvulsive drug gabapentine in the treatment of pain associated with herpes zoster. Methods/Design: Three-armed, randomised, placebo-controlled trial with a total follow-up time of 6 months. Up to estimated 336 patients (interim analyses) with acute herpes zoster pain (VAS > 30 mm) will be randomised to one of three groups (a) semi-standardised acupuncture (168 patients); (b) gabapentine with individualised dosage between 900-3600 mg/d (84 patients); (c) sham laser acupuncture. Intervention takes place over 4 weeks, all patients will receive analgesic therapy (non-opioid analgesics: metamizol or paracetamol and opioids: tramadol or morphine). Therapy phase includes 4 weeks in which group (a) and (c) consist of 12 sessions per patient, (b) visits depend on patients needs. Main outcome measure is to assess the alteration of pain intensity before and 1 week after treatment sessions (visual analogue scale VAS 0-100 mm). Secondary outcome measure are: alteration of pain intensity and frequency of pain attacks; alteration of different aspects of pain evaluated by standardised pain questionnaires (NPI, PDI, SES); effects on quality of life (SF 36); analgesic demand; alteration of sensoric perception by systematic quantitative sensory testing (QST); incidence of postherpetic neuralgia; side effects and cost effectiveness. Credibility of treatments will be assessed. Discussion: This study is the first large-scale randomised placebo controlled trial to evaluate the efficacy of acupuncture compared to gabapentine and sham treatment and will provide valuable new information about the clinical and physiological effects of acupuncture and gabapentine in the treatment of acute herpes zoster pain. The study has been pragmatically designed to ensure that the study findings can be implemented into clinical practice if acupuncture can be shown to be an effective treatment strategy in acute herpes zoster pain

Discrepancy between prevalence and perceived effectiveness of treatment methods in myofascial pain syndrome: Results of a cross-sectional, nationwide survey

Background: Myofascial pain is a common dysfunction with a lifetime prevalence affecting up to 85% of the general population. Current guidelines for the management of myofascial pain are not available. In this study we investigated how physicians on the basis of prescription behaviour evaluate the effectiveness of treatment options in their management of myofascial pain. Methods: We conducted a cross-sectional, nationwide survey with a standardized questionnaire among 332 physicians (79.8% male, 25.6% female, 47.5 +/- 9.6 years) experienced in treating patients with myofascial pain. Recruitment of physicians took place at three German meetings of pain therapists, rheumatologists and orthopaedists, respectively. Physicians estimated the prevalence of myofascial pain amongst patients in their practices, stated what treatments they used routinely and then rated the perceived treatment effectiveness on a six-point scale (with 1 being excellent). Data are expressed as mean +/- standard deviation. Results: The estimated overall prevalence of active myofascial trigger points is 46.1 +/- 27.4%. Frequently prescribed treatments are analgesics, mainly metamizol/paracetamol (91.6%), non-steroidal anti-inflammatory drugs/coxibs (87.0%) or weak opioids (81.8%), and physical therapies, mainly manual therapy (81.1%), TENS (72.9%) or acupuncture (60.2%). Overall effectiveness ratings for analgesics (2.9 +/- 0.7) and physical therapies were moderate (2.5 +/- 0.8). Effectiveness ratings of the various treatment options between specialities were widely variant. 54.3% of all physicians characterized the available treatment options as insufficient. Conclusions: Myofascial pain was estimated a prevalent condition. Despite a variety of commonly prescribed treatments, the moderate effectiveness ratings and the frequent characterizations of the available treatments as insufficient suggest an urgent need for clinical research to establish evidence-based guidelines for the treatment of myofascial pain syndrome

Etoricoxib - preemptive and postoperative analgesia (EPPA) in patients with laparotomy or thoracotomy - design and protocols

<p>Abstract</p> <p>Background and Objective</p> <p>Our objective was to report on the design and essentials of the <it>Etoricoxib </it>protocol<it>- Preemptive and Postoperative Analgesia (EPPA) </it>Trial, investigating whether preemptive analgesia with cox-2 inhibitors is more efficacious than placebo in patients who receive either laparotomy or thoracotomy.</p> <p>Design and Methods</p> <p>The study is a 2 × 2 factorial armed, double blinded, bicentric, randomised placebo-controlled trial comparing (a) etoricoxib and (b) placebo in a pre- and postoperative setting. The total observation period is 6 months. According to a power analysis, 120 patients scheduled for abdominal or thoracic surgery will randomly be allocated to either the preemptive or the postoperative treatment group. These two groups are each divided into two arms. Preemptive group patients receive etoricoxib prior to surgery and either etoricoxib again or placebo postoperatively. Postoperative group patients receive placebo prior to surgery and either placebo again or etoricoxib after surgery (2 × 2 factorial study design). The Main Outcome Measure is the cumulative use of morphine within the first 48 hours after surgery (measured by patient controlled analgesia PCA). Secondary outcome parameters include a broad range of tests including sensoric perception and genetic polymorphisms.</p> <p>Discussion</p> <p>The results of this study will provide information on the analgesic effectiveness of etoricoxib in preemptive analgesia and will give hints on possible preventive effects of persistent pain.</p> <p>Trial registration</p> <p>NCT00716833</p

Impact of Anti-Inflammatory Agents on the Gene Expression Profile of Stimulated Human Neutrophils: Unraveling Endogenous Resolution Pathways

Adenosine, prostaglandin E2, or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A2A receptor agonist CGS 21680, prostaglandin E2, cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution

Lysophosphatides enhance superoxide responses of stimulated human neutrophils

Human neutrophils which are pretreated with subtoxic concentrations of a variety of lysophosphatides (lysophosphatidytcholine, lysophosphatidylcholine oleoyl, lysophosphatidylcholine myrioyl, lysophosphatidylcholine stearoyl, lysophosphatidylcholine gamma- O -hexadecyl, lysophosphatidylinositol, and lysophosphatidylglycerol) act synergistically with neutrophil agonists phorbol myristate acetate, immune complexes, poly- L -histidine, phytohemagglutinin, and N -formyl methionyl-leucyl-phenyalanine to cause enhanced generation of superoxide (O 2 − ). None of the lyso compounds by themselves caused generation of O 2 − . The lyso compounds strongly bound to the neutrophils and could not be washed away. All of the lyso compounds that collaborated with agonists to stimulate O 2 − generation were hemolytic for human red blood cells. On the other hand, lyso compounds that were nonhemolytic for red blood cells (lysophosphatidylcholine caproate, lysophosphatidylcholine decanoyl, lysophosphatidylethanolamine, lysophosphatidylserine) failed to collaborate with agonists to generate synergistic amounts of O 2 − . However, in the presence of cytochalasin B, both lysophosphatidyiethanolamine and lysophosphatidylserine also markedly enhanced O 2 − generation induced by immune complexes. O 2 − generation was also very markedly enhanced when substimulatory amounts of arachidonic acid or eicosapentanoic acid were added to PMNs in the presence of a variety of agonists. On the other hand, neither phospholipase C, streptolysin S (highly hemolytic), phospholipase A 2 , phosphatidylcholine, nor phosphatidylcholine dipalmitoyl (all nonhemolytic) had the capacity to synergize with any of the agonists tested to generate enhanced amounts of O 2 − . The data suggest that in addition to long-chain fatty acids, only those lyso compounds that possess fatty acids with more than 10 carbons and that are also highly hemolytic can cause enhanced generation of O 2 − in stimulated PMNs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44499/1/10753_2004_Article_BF00924787.pd