TY - JOUR A1 - Stange, Katja A1 - Désir, Julie A1 - Kakar, Naseebullah A1 - Mueller, Thomas D. A1 - Budde, Birgit S. A1 - Gordon, Christopher T. A1 - Horn, Denise A1 - Seemann, Petra A1 - Borck, Guntram T1 - A hypomorphic BMPR1B mutation causes du Pan acromesomelic dysplasia JF - Orphanet Journal of Rare Diseases N2 - Background: Grebe dysplasia, Hunter-Thompson dysplasia, and du Pan dysplasia constitute a spectrum of skeletal dysplasias inherited as an autosomal recessive trait characterized by short stature, severe acromesomelic shortening of the limbs, and normal axial skeleton. The majority of patients with these disorders have biallelic loss-of-function mutations of GDF5. In single instances, Grebe dysplasia and a Grebe dysplasia-like phenotype with genital anomalies have been shown to be caused by mutations in BMPR1B, encoding a GDF5 receptor. Methods: We clinically and radiologically characterised an acromesomelic chondrodysplasia in an adult woman born to consanguineous parents. We sequenced GDF5 and BMPR1B on DNA of the proposita. We performed 3D structural analysis and luciferase reporter assays to functionally investigate the identified BMPR1B mutation. Results: We extend the genotype-phenotype correlation in the acromesomelic chondrodysplasias by showing that the milder du Pan dysplasia can be caused by a hypomorphic BMPR1B mutation. We show that the homozygous c.91C>T, p.(Arg31Cys) mutation causing du Pan dysplasia leads to a significant loss of BMPR1B function, but to a lesser extent than the previously reported p.Cys53Arg mutation that results in the more severe Grebe dysplasia. Conclusions: The phenotypic severity gradient of the clinically and radiologically related acromesomelic chondrodysplasia spectrum of skeletal disorders may be due to the extent of functional impairment of the ligand-receptor pair GDF5-BMPR1B. KW - linkage analysis KW - chondrodysplasia KW - specificity KW - Grebe dysplasia KW - BMPR1B KW - du Pan dysplasia KW - tool KW - missense KW - grebe KW - protein-1 CDMP1 gene KW - Acromesomelic dysplasias Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151650 VL - 10 IS - 84 ER - TY - JOUR A1 - Planes, Maria D. A1 - Niñoles, Regina A1 - Rubio, Lourdes A1 - Bissoli, Gaetano A1 - Bueso, Eduardo A1 - García-Sánchez, María J. A1 - Alejandro, Santiago A1 - Gonzalez-Guzmán, Miguel A1 - Hedrich, Rainer A1 - Rodriguez, Pedro L. A1 - Fernández, José A. A1 - Serrano, Ramón T1 - A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane \(H^+\)-ATPase and decreased cytosolic pH, \(K^+\), and anions JF - Journal of Experimental Botany N2 - The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for \(H^+\) efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit \(H^+\) efflux (\(H^+\)-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H+-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (\(H^+\) efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to \(K^+\) efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the \(H^+\)-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of (\(H^+\)-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of (\(H^+\)-ATPase while the guard-cell kinase SnRK2.6/OST1 did not. KW - ABA receptors KW - cytosolic pH KW - ion channels KW - microelectrodes KW - protein kinase KW - proton efflux Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121221 VL - 66 IS - 3 ER - TY - JOUR A1 - Sexauer, Moritz A1 - Bhasin, Hemal A1 - Schön, Maria A1 - Roitsch, Elena A1 - Wall, Caroline A1 - Herzog, Ulrike A1 - Markmann, Katharina T1 - A micro RNA mediates shoot control of root branching JF - Nature Communications N2 - Plants extract mineral nutrients from the soil, or from interactions with mutualistic soil microbes via their root systems. Adapting root architecture to nutrient availability enables efficient resource utilization, particularly in patchy and dynamic environments. Root growth responses to soil nitrogen levels are shoot-mediated, but the identity of shoot-derived mobile signals regulating root growth responses has remained enigmatic. Here we show that a shoot-derived micro RNA, miR2111, systemically steers lateral root initiation and nitrogen responsiveness through its root target TML (TOO MUCH LOVE) in the legume Lotus japonicus, where miR2111 and TML were previously shown to regulate symbiotic infections with nitrogen fixing bacteria. Intriguingly, systemic control of lateral root initiation by miR2111 and TML/HOLT (HOMOLOGUE OF LEGUME TML) was conserved in the nonsymbiotic ruderal Arabidopsis thaliana, which follows a distinct ecological strategy. Thus, the miR2111-TML/HOLT regulon emerges as an essential, conserved factor in adaptive shoot control of root architecture in dicots. KW - evolutionary developmental biology KW - plant development KW - plant molecular biology KW - plant signalling Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357472 VL - 14 ER - TY - JOUR A1 - Palamides, Pia A1 - Jodeleit, Henrika A1 - Föhlinger, Michael A1 - Beigel, Florian A1 - Herbach, Nadja A1 - Mueller, Thomas A1 - Wolf, Eckhard A1 - Siebeck, Matthias A1 - Gropp, Roswitha T1 - A mouse model for ulcerative colitis based on NOD-scid IL2R gamma(null) mice reconstituted with peripheral blood mononuclear cells from affected individuals JF - Disease Models & Mechanisms N2 - Animal models reflective of ulcerative colitis (UC) remain a major challenge, and yet are crucial to understand mechanisms underlying the onset of disease and inflammatory characteristics of relapses and remission. Mouse models in which colitis-like symptoms are induced through challenge with toxins such as oxazolone, dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) have been instrumental in understanding the inflammatory processes of UC. However, these neither reflect the heterogeneous symptoms observed in the UC-affected population nor can they be used to test the efficacy of inhibitors developed against human targets where high sequence and structural similarity of the respective ligands is lacking. In an attempt to overcome these problems, we have developed a mouse model that relies on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells derived from UC-affected individuals. Upon challenge with ethanol, mice developed colitis-like symptoms and changes in the colon architecture, characterized by influx of inflammatory cells, edema, crypt loss, crypt abscesses and epithelial hyperplasia, as previously observed in immune-competent mice. TARC, TGFβ1 and HGF expression increased in distal parts of the colon. Analysis of human leucocytes isolated from mouse spleen revealed an increase in frequencies of CD1a+, CD64+, CD163+ and TSLPR+ CD14+ monocytes, and antigen-experienced CD44+ CD4+ and CD8+ T-cells in response to ethanol. Analysis of human leucocytes from the colon of challenged mice identified CD14+ monocytes and CD11b+ monocytes as the predominant populations. Quantitative real-time PCR (RT-PCR) analysis from distal parts of the colon indicated that IFNγ might be one of the cytokines driving inflammation. Treatment with infliximab ameliorated symptoms and pathological manifestations, whereas pitrakinra had no therapeutic benefit. Thus, this model is partially reflective of the human disease and might help to increase the translation of animal and clinical studies. KW - animal models KW - Ulcerative colitis KW - NSG mice KW - Infliximab KW - Pitrakinra Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164946 VL - 9 ER - TY - JOUR A1 - Jahn, Martin T. A1 - Schmidt, Katrin A1 - Mock, Thomas T1 - A novel cost effective and high-throughput isolation and identification method for marine microalgae JF - Plant Methods N2 - BACKROUND: Marine microalgae are of major ecologic and emerging economic importance. Biotechnological screening schemes of microalgae for specific traits and laboratory experiments to advance our knowledge on algal biology and evolution strongly benefit from culture collections reflecting a maximum of the natural inter- and intraspecific diversity. However, standard procedures for strain isolation and identification, namely DNA extraction, purification, amplification, sequencing and taxonomic identification still include considerable constraints increasing the time required to establish new cultures. RESULTS: In this study, we report a cost effective and high-throughput isolation and identification method for marine microalgae. The throughput was increased by applying strain isolation on plates and taxonomic identification by direct PCR (dPCR) of phylogenetic marker genes in combination with a novel sequencing electropherogram based screening method to assess the taxonomic diversity and identity of the isolated cultures. For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean. These cultures include the isolate of a novel marine Chlorophyceae strain among several different diatoms. CONCLUSIONS: We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks. Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures." KW - cultivation KW - direct PCR KW - isolation KW - marine microalgae KW - taxonomy Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121255 VL - 10 IS - 26 ER - TY - JOUR A1 - Shepard, Blythe D. A1 - Cheval, Lydie A1 - Peterlin, Zita A1 - Firestein, Stuart A1 - Koepsell, Hermann A1 - Doucet, Alain A1 - Pluznick, Jennifer L. T1 - A Renal Olfactory Receptor Aids in Kidney Glucose Handling JF - Scientific Reports N2 - Olfactory receptors (ORs) are G protein-coupled receptors which serve important sensory functions beyond their role as odorant detectors in the olfactory epithelium. Here we describe a novel role for one of these ORs, Olfr1393, as a regulator of renal glucose handling. Olfr1393 is specifically expressed in the kidney proximal tubule, which is the site of renal glucose reabsorption. Olfr1393 knockout mice exhibit urinary glucose wasting and improved glucose tolerance, despite euglycemia and normal insulin levels. Consistent with this phenotype, Olfr1393 knockout mice have a significant decrease in luminal expression of Sglt1, a key renal glucose transporter, uncovering a novel regulatory pathway involving Olfr1393 and Sglt1. In addition, by utilizing a large scale screen of over 1400 chemicals we reveal the ligand profile of Olfr1393 for the first time, offering new insight into potential pathways of physiological regulation for this novel signaling pathway. KW - olfactory receptor KW - Olfr1393 KW - kidney KW - glucose handling Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167605 VL - 6 IS - 35215 ER - TY - JOUR A1 - Harth, Stefan A1 - Kotzsch, Alexander A1 - Hu, Junli A1 - Sebald, Walter A1 - Mueller, Thomas D. T1 - A Selection Fit Mechanism in BMP Receptor IA as a Possible Source for BMP Ligand-Receptor Promiscuity N2 - Background: Members of the TGF-b superfamily are characterized by a highly promiscuous ligand-receptor interaction as is readily apparent from the numeral discrepancy of only seven type I and five type II receptors available for more than 40 ligands. Structural and functional studies have been used to address the question of how specific signals can be deduced from a limited number of receptor combinations and to unravel the molecular mechanisms underlying the protein-protein recognition that allow such limited specificity. Principal Findings: In this study we have investigated how an antigen binding antibody fragment (Fab) raised against the extracellular domain of the BMP receptor type IA (BMPR-IA) recognizes the receptor’s BMP-2 binding epitope and thereby neutralizes BMP-2 receptor activation. The crystal structure of the complex of the BMPR-IA ectodomain bound to the Fab AbD1556 revealed that the contact surface of BMPR-IA overlaps extensively with the contact surface for BMP-2 interaction. Although the structural epitopes of BMPR-IA to both binding partners coincides, the structures of BMPR-IA in the two complexes differ significantly. In contrast to the structural differences, alanine-scanning mutagenesis of BMPR-IA showed that the functional determinants for binding to the antibody and BMP-2 are almost identical. Conclusions: Comparing the structures of BMPR-IA bound to BMP-2 or bound to the Fab AbD1556 with the structure of unbound BMPR-IA shows that binding of BMPR-IA to its interaction partners follows a selection fit mechanism, possibly indicating that the ligand promiscuity of BMPR-IA is inherently encoded by structural adaptability. The functional and structural analysis of the BMPR-IA binding antibody AbD1556 mimicking the BMP-2 binding epitope may thus pave the way for the design of low-molecular weight synthetic receptor binders/inhibitors. KW - Physiologische Chemie KW - antigen binding antibody fragment (Fab) Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68497 ER - TY - JOUR A1 - Dindas, Julian A1 - Dreyer, Ingo A1 - Huang, Shouguang A1 - Hedrich, Rainer A1 - Roelfsema, M. Rob G. T1 - A voltage-dependent Ca\(^{2+}\) homeostat operates in the plant vacuolar membrane JF - New Phytologist N2 - Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca\(^{2+}\) signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca\(^{2+}\) and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca\(^{2+}\) concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H\(^{+}\)/Ca\(^{2+}\) exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca\(^{2+}\) homeostat could contribute to calcium signalling when coupled to a recently discovered K\(^{+}\) channel-dependent module for electrical excitability of the vacuolar membrane. KW - voltage clamp KW - Arabidopsis thaliana KW - calcium signalling KW - computational cell biology KW - cpYFP cytosolic pH reporter KW - R-GECO1 cytosolic Ca\(^{2+}\) reporter KW - TPC1 channel KW - vacuolar membrane Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259627 VL - 230 IS - 4 ER - TY - THES A1 - Nhan, Pham Phuoc T1 - Accumulation and biological activity of oxidized lipids in Anabaena PCC 7120 T1 - Akkumulation und biologische Aktivität von oxidierten Fettsäuren in Anabaena PCC 7120 N2 - Oxylipine sind wichtige biologisch aktive Verbindungen, die entscheidende Rollen in der Abwehr, dem Wachstum, der Entwicklung und der Reproduktion von Pflanzen und Tieren spielen. Oxylipine können entweder über enzymatische Wege oder eine Radikal-katalysierte Reaktion gebildet werden. Enzymatische und nicht-enzymatische Oxidationsprodukte der Arachidonsäure (C20:4) in Tieren sind Prostaglandine und Isoprostane. In Pflanzen werden ausgehend von der -Linolensäure (C18:3) über einen enzymatischen Weg OPDA und Jasmonsäure und durch Radikal-katalysierte Reaktion Phytoprostane gebildet. Die Membranen von Cyanobakterien enthalten, ähnlich denen von Pflanzen, einen großen Anteil an mehrfach ungesättigten Fettsäuren, ca. 25% der gesamten Fettsäuren. Biosynthese und Funktionen der Oxylipine wurden an zwei Modell-Cyanobakterien, Anabaena PCC 7120 und Synechocystis PCC 6803 untersucht: 1. Das fadenförmige Cyanobakterium Anabaena PCC 7120 kann Phytoprostane Typ I und II sowie Hydroxyfettsäuren ähnlich wie Pflanzen produzieren aber die enzymatische Ausstattung zur Bildung von Jasmonaten (12-oxo-Phytodiensäure und Jasmonsäure) und Prostaglandinen ist nicht vorhanden. Die erhaltenen Daten stellen den ersten Nachweis für das Vorkommen von Phytoprostanen in Cyanobakterien bzw. bei Prokaryonten dar. 2. Durch GC-MS Analyse wurden E1- and F1-Phytoprostane in Anabaena PCC 7120 in freier und veresterter Form detektiert. Die Spiegel sind vergleichbar mit denen in Pflanzen und lagen im Bereich von ng/g TG. PPF1 ließen sich nicht in einwöchigen Kulturen nachweisen, die Spiegel in sechswöchigen Kulturen lagen bei 142 ng/l. Die Spiegel von PPE1 waren hingegen in ein- und sechswöchigen Kulturen ähnlich und lagen bei ca. 20 ng/g TG. Die Mengen an freien PPE1 in den Zellen waren mit 80.5  23.6 etwa viermal höher als die von PPF1 mit 24.1  10.9 ng/g TG. Allerdings gab es keine signifikanten Unterschiede in den Spiegeln an gesamten PPF1 und PPE1 in den Zellen, sie lagen im Bereich von 150 bis zu ca. 200 ng/g TG. 3. Die Akkumulation von Phytoprostanen in Anabaena ist induzierbar. Nach der Kombination von oxidativem Stress (200 µM H2O2 oder 10 µM CuSO4) und hoher Lichtintensität (330 µE.m-2.s-1) für 8 h stiegen die Spiegel an gesamten PPE1 und PPF1 um den Faktor 2 bis 4 an. Interessanterweise führte im Gegensatz zu höheren Pflanzen die Applikation von oxidativem Stress oder hoher Lichtintensität alleine nicht zur Induktion der Phytoprostanakkumulation in diesen Cyanobakterien. 4. Eine Vorbehandlung von Anabaena Zellen mit exogenen Phytoprostanen führte zu einer erhöhten Toleranz gegenüber oxidativem Stress. Alle Phytoprostane außer PPE1 zeigten einen Schutzeffekt. Eine Mischung von PPA1 Typ I und II ergab den höchsten Schutzeffekt. Eine Vorinkubation von Anabena Zellen mit 100 µM PPA1–type I/II für 16 h schützte 84.2% beziehungsweise 77.5% der Zellen vor einer anschießenden lethalen Applikation von 1 mM H2O2 beziehungsweise 50 µM CuSO4 für 5 h. Ohne eine Oxylipin-Vorinkubation starben etwa 98% der Zellen. Überraschenderweise ergab auch die Vorbehandlung mit anderen, enzymatisch gebildeten Oxylipinen aus Tieren und Pflanzen einen Schutzeffekt, der allerdings nur 10 bis 30% betrug. Dagegen schützte eine Phytoprostan-Vorbehandlung nicht Pseudomonas syringae und Escherichia coli gegen toxische Mengen von Wasserstoffperoxid. Allerdings fehlen in den Membranen dieser Bakterien mehrfach ungesättigte Fettsäuren und deshalb endogen oxydierte Lipide. 5. Eine exogene Applikation von 100 µM PPF1 oder 1,5 mM H2O2 führte in Anabaena nicht zu einer Induktion der Expression des isiA Gens. Oxylipin-Behandlungen zeigten auch keine Wirkung auf Shinorin- und Tocopherol-Spiegel in Anabaena. Die Applikation von 100 µM PPF1 für 6 h führte aber zu Änderungen im Proteinmuster in Anabaena. Der größte Teil der differentiellen Proteine wurde durch PPF1 herunterreguliert. Bei vielen dieser Proteine handelt es sich um photosynthetische Proteine. Da ein oxidativer Stress nur in der Kombination mit hoher Lichtintensität die Lipidperoxidation erhöht, könnte die negative Regulation der Photosynthese nach Erkennung von oxydierten Lipiden (Phytoprostanen) eine Überlebens-Strategie sein um Schäden durch peroxidierte Lipide zu vermeiden. 6. Tote Pflanzen könnten eine hauptsächliche Quelle der exogenen Phytoprostane in der natürlichen Umgebung von Anabaena sein. Trockenes Heu gibt PPE1 und PPF1 (11 µg/g TG) an die wässerige Umgebung ab. Anabaena ist ein typisches Cyanobakterium in Reisfeldern. Nach der Ernte bleiben meist die nicht genutzten Teile der Reispflanzen auf dem Feld. Diese könnten Phytoprostane abgeben, die wiederum einen Einfluß auf die Cyanobakterien im Reis-Ökosystem haben könnten. 7. Eine neue Kategorie von Oxylipinen, die Phytoprostane Typ III und IV, wurden in vitro identifiziert und quantifiziert. Die beiden Haupt-Phytoprostane, PPE1 und PPF1 (Typ III und IV), können durch die Autoxidation der -Linolensäure oder des Borretschsamenöls (enthält 25% der -Linolensäure) gewonnen werden. Nach 12 Tagen Autoxidation und anschließender Hydrolyse wurden aus 1 g Borretschsamenöl 112,71 ± 1,93 µg PPF1 und 3,80 ± 0,14 mg PPE1 isoliert. PPB1 und PPA1 (Typ III und IV) wurden durch Isomerisierung und Dehydratisierung von PPE1 hergestellt. Die Ausbeute von PPB1 lag bei 1,71 ± 0,04 mg/g Öl (Typ III) und 2,09 ± 0,12 mg/g Öl (Typ IV), die von PPA1 lag bei 8,38 ± 0,35 µg/g und 10,18 ± 0,30 µg/g Öl. 8. Es wurde eine schnelle HPLC-MS/MS Methode für die Analytik der Phytoprostane und Phytohormone entwickelt. Diese Methode wurde für die Quantifizierung von freien und veresterten E1- and F1-Phytoprostane Typ III und IV in Synechocystis PCC 6803 angewendet. Die Phytoprostane Typ III und IV sind in vivo in freier und veresterter Form vorhanden. Die Spiegel der gesamten PPE1 Typ III und IV in Synechocystis sind mindestens doppelt so hoch wie die von PPF1. Im Gegensatz zu Anabaena, waren PPE1 und PPF1 in ein- und sechswöchigen Kulturen von Synechocystis detektierbar. Die Spiegel an freien PPF1 im Medium (231,8 ± 36,2 ng/l) und in den Zellen (164,9 ± 15,2 ng/g TG) waren niedriger als die von PPE1 (1003,3 ± 365,2 ng/l und 2331,0 ± 87,7 ng/g TG). N2 - Oxylipins are important biological active compounds that play essential roles in defense, growth, development, and reproduction of plants and animals. Oxylipins are formed either by enzymatic pathways or radical catalyzed reaction from polyunsaturated fatty acids. Products of oxidation of arachidonic acid (C20:4) in animals by enzymatic and non-enzymatic pathways are prostaglandins and isoprostanes, respectively. In plants, radical catalyzed reaction of -linolenic acid (C18:3) forms phytoprostanes and enzymatic oxidation of this fatty acid produces OPDA and jasmonic acid. Like plants, cyanobacterial membranes contain a high ratio of polyunsaturated fatty acid, about 25% of total fatty acids. Oxylipin biosynthesis and function was studied in two model cyanobacteria, Anabaena PCC 7120 and Synechocystis PCC 6803, for the first time: 1. The filamentous cyanobaterium Anabaena PCC 7120 can naturally produce phytoprostanes type I and II as well as hydroxy fatty acids like in plants but lacks the enzymatic capacity to form jasmonates (12-oxo-phytodienoic acid and jasmonic acid) and prostaglandins. Data obtained provide the first evidence for the occurence of phytoprostanes in cyanobacteria as well as in the baterial kingdom. 2. By GC-MS analysis, the E1- and F1-phytoprostanes in Anabaena PCC 7120 were detected both in free and esterified form. Their levels are comparable with those in plants, in the range of ng/g DW. In one week old cultures, there was no evidence of PPF1 in the medium but its level accumulated up to 142 ng/l in six weeks old cultures. In contrast, PPE1 was stable over time, about 20 ng/g DW. Free cellular PPE1 was found about 4 times higher than that of PPF1, 80.5  23.6 and 24.1  10.9 ng/g DW, respectively. However, there was no significant difference in the total cellular levels of PPF1 and PPE1, ranging from 150 to about 200 ng/g DW. 3. Phytoprostanes are inducible in Anabaena. In the combination of oxidative stress (200 µM H2O2 or 10 µM CuSO4) with high light intensity (330 µE.m-2.s-1) for 8 h, levels of total cellular PPE1 and PPF1 were increased about 2 to 4 times. Interestingly, unlike in higher plants, application of oxidative stress or high light intensity alone showed no phytoprostaneous induction in this cyanobacterium. 4. When Anabaena cells were treated with phytoprostanes, Anabaena cells became remarkably resistant against subsequently applied – otherwise lethal – oxidative stress. All phytoprostanes displayed a high protective effect except for PPE1. The highest protection level was contributed by a mixture of PPA1 type I and II. After preincubation of Anabena cells with 100 µM PPA1–type I/II for 16 h followed by application of 1 mM H2O2 or 50 µM CuSO4 for 5 h, A1-phytoprostane pre-treatment protected 84.2% and 77.5% of the cells from cell death, respectively. Without oxylipins pre-treatment, about 98% of the cells were dead. Surprisingly, preincubation of Anabaena with other oxylipins derived from enzymatic pathway in plants and animals showed also an effect, however, the protection effect was low and ranged from 10 to 30%. In contrast, phytoprostanes did not protect Pseudomonas syringae and Escherichia coli from the toxicity of hydrogen peroxide. However, these bacteria do not synthesize polyunsaturated fatty acids and are therefore devoid of and not exposed to endogenously formed oxidized lipids. 5. Exogenous application of 100 µM PPF1 or 1.5 mM H2O2 for 90 min did not activate the expression of isiA in Anabaena. Oxylipins also displayed no effect on shinorine and tocopherol levels in Anabaena. However, application of 100 µM PPF1 for 6 h altered the protein expression in Anabaena. Most PPF1-modulated proteins are down-regulated and related to photosynthesis. Since oxidative stress only in combination with high light intensity increased lipid peroxidation, down-regulation of photosynthesis after recognition of oxidised lipids (phytoprostanes) may be a survival strategy of Anabaena to avoid damage by peroxidized lipids. 6. Dead plants may be the main source of (exogenous) phytoprostanes in the natural environment of Anabaena. Dry hay releases PPE1 and PPF1 (11 µg/g DW) into an aqueous environment. Anabaena is the typical cyanobacterium in paddy rice fields. After harvesting, most of uneconomical parts of rice plants are abundant on the field, which may release phytoprostanes that in turn might have an impact on cyanobacteria in the rice ecosystems. However, field research is needed to clarify this suspection. 7. A new class of oxylipins, phytoprostanes type III and IV, was identified and quantified in vitro. The two main phytoprostanes, PPE1 and PPF1 (type III and IV), can be obtained by autoxidation of -linolenic acid or Borage oil (containing 25% esterified -linolenic acid). After 12 days of autoxidation and subsequent hydrolysis, 1 g of Borage oil yielded 112.71 ± 1.93 µg of PPF1 and 3.80 ± 0.14 mg of PPE1. PPB1 and PPA1 (type III and IV) were prepared by isomerization and dehydration of PPE1 (type III and IV). The overall yield of PPB1 was 1.71 ± 0.04 mg/g oil (type III) and 2.09 ± 0.12 mg/g oil (type IV). Those of PPA1 were 8.38 ± 0.35 µg/g and 10.18 ± 0.30 µg/oil, respectively. 8. A rapid HPLC-MS/MS method for phytoprostane and phytohormone analysis has been developed. This method was applied to quantify free and esterified E1- and F1-phytoprostanes type III and IV in Synechocystis PCC 6803. The in vivo phytoprostanes type III and IV are present both in free and esterified form. The total cellular level of PPE1 type III and IV in Synechocystis is at least 2 times higher than that of PPF1. Unlike Anabaena, PPE1 and PPF1 were detectable in the medium of one week old Synechocystis cultures. Free levels of PPF1 in the medium (231.8 ± 36.2 ng/l) and in the cells (164.9 ± 15.2 ng/g DW) are lower than those of PPE1 (1003.3 ± 365.2 ng/l and 2331.0 ± 87.7 ng/g DW). KW - Oxidativer Stress KW - oxidative stress KW - phytoprostane KW - oxidized lipids Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-24347 ER - TY - JOUR A1 - Abdelmohsen, Usama Ramadan A1 - Yang, Chen A1 - Horn, Hannes A1 - Hajjar, Dina A1 - Ravasi, Timothy A1 - Hentschel, Ute T1 - Actinomycetes from Red Sea Sponges: Sources for Chemical and Phylogenetic Diversity N2 - The diversity of actinomycetes associated with marine sponges collected off Fsar Reef (Saudi Arabia) was investigated in the present study. Forty-seven actinomycetes were cultivated and phylogenetically identified based on 16S rRNA gene sequencing and were assigned to 10 different actinomycete genera. Eight putatively novel species belonging to genera Kocuria, Mycobacterium, Nocardia, and Rhodococcus were identified based on sequence similarity values below 98.2% to other 16S rRNA gene sequences available in the NCBI database. PCR-based screening for biosynthetic genes including type I and type II polyketide synthases (PKS-I, PKS-II) as well as nonribosomal peptide synthetases (NRPS) showed that 20 actinomycete isolates encoded each at least one type of biosynthetic gene. The organic extracts of nine isolates displayed bioactivity against at least one of the test pathogens, which were Gram-positive and Gram-negative bacteria, fungi, human parasites, as well as in a West Nile Virus protease enzymatic assay. These results emphasize that marine sponges are a prolific resource for novel bioactive actinomycetes with potential for drug discovery. KW - PKS I KW - Meeresschwämme KW - PKS II KW - NRPS KW - Red sea KW - sponges KW - actinomycetes KW - bioactivity Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112882 ER -