phosphoinositide 3 kinase gamma controls inflammation induced myocardial depression via sequential camp and inos signalling

Aims Sepsis-induced myocardial depression (SIMD), an early and frequent event of infection-induced systemic inflammatory response syndrome (SIRS), is characterized by reduced contractility irrespective of enhanced adrenergic stimulation. Phosphoinositide-3 kinase γ (PI3Kγ) is known to prevent β-adrenergic overstimulation via its scaffold function by activating major cardiac phosphodiesterases and restricting cAMP levels. However, the role of PI3Kγ in SIRS-induced myocardial depression is unknown. This study is aimed at determining the specific role of lipid kinase-dependent and -independent functions of PI3Kγ in the pathogenesis of SIRS-induced myocardial depression. Methods and results PI3Kγ knockout mice (PI3Kγ−/−), mice expressing catalytically inactive PI3Kγ (PI3KγKD/KD), and wild-type mice (P3Kγ+/+) were exposed to lipopolysaccharide (LPS)-induced systemic inflammation and assessed for survival, cardiac autonomic nervous system function, and left ventricular performance. Additionally, primary adult cardiomyocytes were used to analyse PI3Kγ effects on myocardial contractility and inflammatory response. SIRS-induced adrenergic overstimulation induced a transient hypercontractility state in PI3Kγ−/− mice, followed by reduced contractility. In contrast, P3Kγ+/+ mice and PI3KγKD/KD mice developed an early and ongoing myocardial depression despite exposure to similarly increased catecholamine levels. Compared with cells from P3Kγ+/+ and PI3KγKD/KD mice, cardiomyocytes from PI3Kγ−/− mice showed an enhanced and prolonged cAMP-mediated signalling upon norepinephrine and an intensified LPS-induced proinflammatory response characterized by nuclear factor of activated T-cells-mediated inducible nitric oxide synthase up-regulation. Conclusions This study reveals the lipid kinase-independent scaffold function of PI3Kγ as a mediator of SIMD during inflammation-induced SIRS. Activation of cardiac phosphodiesterases via PI3Kγ is shown to restrict myocardial hypercontractility early after SIRS induction as well as the subsequent inflammatory responses

Biosynthesis of allene oxides in Physcomitrella patens

Background: The moss Physcomitrella patens contains C-18- as well as C-20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2. Results: Our results show that PpAOS1 is highly active with both C-18 and C-20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C-20-substrates, exhibiting trace activity (similar to 1000-fold lower k(cat)/K-M) with C-18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis. Conclusions: Our study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C-18- and C-20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes

Glioblastoma cells express functional cell membrane receptors activated by daily used medical drugs

PURPOSE: Calcium ions are highly versatile spacial and temporal intracellular signals of non-excitable cells and have an important impact on nearly every aspect of cellular life controlling cell growth, metabolism, fluid secretion, information processing, transcription, apoptosis, and motility. Neurons and glia respond to stimuli, including neurotransmitters, neuromodulators, and hormones, which increase the intracellular calcium concentration. The function of intracellular calcium in gliomas is unknown. Lots of daily used drugs may act via receptors that can be linked to the intracellular calcium system and therefore could influence glioma biology. METHODS: Glioma cells were loaded with the calcium ion sensitive dye Fura 2-AM. Subsequently, cells were stimulated with 25 different medical drugs for 30 s. The increase of free intracellular calcium ions was measured and calculated by a microscope–camera–computer-unit. RESULTS: Except for the buffer solution HEPES that served as negative control and for the cortisol derivative dexamethasone, all other 24 tested drugs induced a rise of intracellular calcium ions. The cellular calcium responses were classified into seven functional groups. The tested substances activated several types of calcium channels and receptors. CONCLUSIONS: Our study impressively demonstrates that medical drugs are potent inducers of intracellular calcium signals. Totally unexpected, the results show a high amount of functional cellular receptors and channels on glioma cells, which could be responsible for certain biological effects like migration and cell growth. This calcium imaging study proves the usability of the calcium imaging as a screening system for functional receptors on human glioma cells

Genomic Analysis of the Hydrocarbon-Producing, Cellulolytic, Endophytic Fungus Ascocoryne sarcoides

The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Endophytes represent a promising group of organisms, as they are a mostly untapped reservoir of metabolic diversity. They are often able to degrade cellulose, and they can produce an extraordinary diversity of metabolites. The filamentous fungal endophyte Ascocoryne sarcoides was shown to produce potential-biofuel metabolites when grown on a cellulose-based medium; however, the genetic pathways needed for this production are unknown and the lack of genetic tools makes traditional reverse genetics difficult. We present the genomic characterization of A. sarcoides and use transcriptomic and metabolomic data to describe the genes involved in cellulose degradation and to provide hypotheses for the biofuel production pathways. In total, almost 80 biosynthetic clusters were identified, including several previously found only in plants. Additionally, many transcriptionally active regions outside of genes showed condition-specific expression, offering more evidence for the role of long non-coding RNA in gene regulation. This is one of the highest quality fungal genomes and, to our knowledge, the only thoroughly annotated and transcriptionally profiled fungal endophyte genome currently available. The analyses and datasets contribute to the study of cellulose degradation and biofuel production and provide the genomic foundation for the study of a model endophyte system

Befragung der Gesundheitsämter zur Umsetzung des Meldewesens nach dem Infektionsschutzgesetz

Die Einführung des Infektionsschutzgesetzes im Januar 2001 hatte inhaltliche und verfahrenstechnische Änderungen in der infektionsepidemiologischen Überwachung meldepflichtiger Erkrankungen in Deutschland zur Folge. Die Umsetzung des Meldesystems wurde mit dem Ziel untersucht, Verbesserungsmöglichkeiten zu identifizieren. Mittels Befragung aller bundesdeutschen Gesundheitsämter wurden folgende Kriterien analysiert: Ressourcen (personelle und technische Ausstattung), Informationsbedarf (Zufriedenheit mit bestehenden Angeboten/weiterer Trainingsbedarf), Datennutzung (Umfang eigener Datenauswertungen/Ergebnisrückkopplung), Akzeptanz (Falldefinitionen/EDV-gestütztes Meldeverfahren). Bezogen auf die Gesamtmitarbeiterzahlen ist ein Anteil von durchschnittlich 11% im Meldewesen tätig. Die Bearbeitung von Meldedaten erfolgt hauptsächlich durch nichtärztliches Personal (78,4%). Fast jedem Mitarbeiter steht dafür ein PC zur Verfügung. Knapp ein Drittel der Gesundheitsämter verwendet das vom RKI entwickelte Meldeprogramm "SurvNet@rki", alle anderen nutzen eine kommerzielle Software. Ihre Erfahrungen mit der EDV bezeichneten 47,1% der Gesundheitsämter als sehr gut bzw. gut. Befriedigend gaben 44,5% an, problematisch 8,4%. Der überwiegende Teil der Gesundheitsämter ist mit den Informationsangeboten des RKI (96,4%) bzw. ihrer Landesstelle (83,7%) zufrieden, dennoch besteht bei 49,1% weiterer Fortbildungsbedarf. Die Verwendung von Falldefinitionen befürworten 95% aller Gesundheitsämter, allerdings werden die Übermittlungskriterien kritisch beurteilt. Die Umsetzung des neuen Meldewesens ist erfolgreich verlaufen. Zur Optimierung des Systems können folgende Maßnahmen beitragen: Vermeidung Software-bedingter Probleme bei der Datenverarbeitung und Übermittlung, Ausbau der vorhandenen Informationsangebote und Bereitstellung zielgruppenorientierter Fortbildungsprogramme sowie Überarbeitung der Falldefinitionen bezüglich einer einfacheren Handhabung.The Infectious Disease Control Act enacted in Germany in January 2001 led to the establishment of a new reporting system for infectious disease. The implementation of this system was evaluated to identify opportunities for further improvement. In a survey of all German local health departments the following criteria were analyzed: resources (staff and technical equipment), information needs (satisfaction with current offers/further training requirements), data analysis (extent of local data analysis/feedback of national data), and acceptability (case definitions/electronic reporting). In local health departments, 11% of the staff were assigned to the infectious disease reporting system. Data were processed mainly by nonmedical staff (78.4%). A computer work-station is available for most staff members. One-third of the local health departments uses the RKI software "SurvNet@rki" for data transmission. All others use commercial software. Experience with the electronic reporting system was rated as very good/good by 47.1% of local health departments, as satisfactory by 44.5%, and as problematic by 8.4%. Most of the local health departments were satisfied with the offers of information provided by RKI (96.4%) and state health departments (83.7%), respectively. However, 49.1% of the local health departments saw a need for further education and training. The implementation of case definitions was supported by 95% of the local health departments, but transmission criteria were criticized. In summary, the new infectious disease reporting system in Germany was successfully implemented. However, the system could be improved through reduction of software problems concerning electronic data processing and transmission, expansion of current offers of in-formation and provision of special staff training programs, and revision of the case definitions concerning clearness and simpler handling.The Infectious Disease Control Act enacted in Germany in January 2001 led to the establishment of a new reporting system for infectious disease. The implementation of this system was evaluated to identify opportunities for further improvement. In a survey of all German local health departments the following criteria were analyzed: resources (staff and technical equipment), information needs (satisfaction with current offers/further training requirements), data analysis (extent of local data analysis/feedback of national data), and acceptability (case definitions/electronic reporting). In local health departments, 11% of the staff were assigned to the infectious disease reporting system. Data were processed mainly by nonmedical staff (78.4%). A computer work-station is available for most staff members. One-third of the local health departments uses the RKI software ldquoSurvNet@rkirdquo for data transmission. All others use commercial software. Experience with the electronic reporting system was rated as very good/good by 47.1% of local health departments, as satisfactory by 44.5%, and as problematic by 8.4%. Most of the local health departments were satisfied with the offers of information provided by RKI (96.4%) and state health departments (83.7%), respectively. However, 49.1% of the local health departments saw a need for further education and training. The implementation of case definitions was supported by 95% of the local health departments, but transmission criteria were criticized. In summary, the new infectious disease reporting system in Germany was successfully implemented. However, the system could be improved through reduction of software problems concerning electronic data processing and transmission, expansion of current offers of in-formation and provision of special staff training programs, and revision of the case definitions concerning clearness and simpler handling

Influenzasurveillance in Deutschland

Das Sentinelsystem der Arbeitsgemeinschaft Influenza (AGI) hat seit 1992 wertvolle Daten zur Influenzaaktivität in der gesamten Bundesrepublik Deutschland gesammelt. Die dabei erfassten Daten zu akuten respiratorischen Erkrankungen bilden die Grundlage für die Bewertung der aktuellen Verbreitung der Influenza. Mit dem Inkrafttreten des Infektionsschutzgesetzes (IfSG) am 1.1.2001 wurde in Deutschland ein neues Meldesystem etabliert. Dabei ist erstmals auch der Nachweis von Influenzaviren meldepflichtig, und zwar über den Meldeweg nach §7.1 IfSG (vorgegebene maximale Übermittlungszeit von 17 Tagen vom Gesundheitsamt bis zum Robert Koch-Institut (RKI)) sowie über eine Schnellübermittlung nach §12 IfSG (vorgegebene Übermittlungszeit von einem Tag). Wir analysierten die Elemente und Inhalte dieses neu angelaufenen Meldesystems und verglichen die Ergebnisse mit denen des AGI-Sentinels. Die Meldedaten nach §12 und die Daten des AGI-Sentinels beschrieben den Verlauf der Influenzawelle übereinstimmend. Insgesamt hat das neue Meldewesen seine Funktionstüchtigkeit eindrucksvoll unter Beweis gestellt.Until recently, influenza surveillance in Germany was conducted through a physician-based sentinel system, the “Arbeitsgemeinschaft Influenza” (AGI), where acute respiratory illnesses are recorded. With the institution of the Protection against Infection Act (IfSG) in January 2001, the laboratory diagnosis of influenza has become notifiable via two different reporting lines: according to § 7.1 IfSG, reporting from the local health department to the Robert Koch-Institute must occur within a maximum of 17 days while rapid reporting according to § 12 IfSG must occur within one day. We analyzed data of the new surveillance system and compared the results with those of the AGI. The epidemic influenza curve according to § 12 was comparable with that of the AGI sentinel system. Overall, the new reporting system has proven its functional capability

Biosynthesis of allene oxides in Physcomitrella patens

Background: The moss Physcomitrella patens contains C-18- as well as C-20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2. Results: Our results show that PpAOS1 is highly active with both C-18 and C-20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C-20-substrates, exhibiting trace activity (similar to 1000-fold lower k(cat)/K-M) with C-18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis. Conclusions: Our study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C-18- and C-20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes