Evolution of specifier proteins in the order Brassicales

Das Glucosinolat-Myrosinase-System ist ein pflanzliches Abwehrsystem der Brassicales. Spezifizierende Proteine sind Teil dieses aktivierten chemischen Abwehrsystems. Bei Gewebeverletzung kommt es zur Hydrolyse der Glucosinolate durch Myrosinasen. Die entstehenden Hydrolyseprodukte lagern sich entweder spontan in toxische, als Abwehrstoffe dienende Isothiocyanate um oder werden durch spezifizierende Proteine in Epithionitrile, einfache Nitrile oder organische Thiocyanate mit weitgehend unbekannter biologischer Funktion umgewandelt. Ziel dieser Arbeit war die Klonierung, Charakterisierung und phylogenetische Analyse spezifizierender Proteine unterschiedlicher Produktspezifität. Durch ein phytochemisches Screening konnten von Isothiocyanaten abweichende Hydrolyseprodukte in 14 Spezies der Brassicaceae sowie in einer Spezies der Tropaeolaceae nachgewiesen werden. Ergänzend zu acht bekannten spezifizierenden Proteinen konnten Full-length cDNAs von drei NSPs, sechs ESPs und zwei TFPs isoliert und die codierten Proteine biochemisch charakterisiert werden. Für die phylogenetischen Analysen standen unter Einbeziehung von drei einer Datenbankrecherche entnommenen und nicht charakterisierten Proteinen 22 Full-length cDNAs und die homologe Sequenz At3g07720 aus A. thaliana zur Verfügung. Die phylogenetischen Stammbäume wurden auf Basis von Nukleotid? und Aminosäuresequenzen mittels verschiedener Verfahren erstellt. Die grundlegend identische Topologie der ermittelten phylogenetischen Stammbäume sowie die Berechnungen von Datenmatritzen für Sequenzdivergenzen und -identitäten als Maß evolutionärer Distanz und die strukturellen Merkmale der spezifizierenden Proteine bestätigen At3g07720 als Vertreter einer Gruppe von Vorläuferproteinen der spezifizierenden Proteine. Ausgehend von At3g07720 und homologen Sequenzen entwickelten sich die NSPs des AtNSP5-Clusters. Eine Aufsplittung dieser NSPs führte zu den NSPs des AtNSP1-Clusters. Eine Duplikation der NSPs des AtNSP1-Clusters führte dann zu den ESPs des ESP/TFP-Clusters. Die Auffächerung der ESPs konnte auf einen Zeitpunkt nach der Auffächerung der Kerngruppe der Brassicaceae in die Abstammungslinien I und II geschätzt und die vorausgehende Genduplikation auf ein Alter von mindestens 35,6 Mill. Jahre datiert werden. Die weitere Evolution ergab eine zweifach unabhängige Entstehung von TFPs ausgehend von ESPs, welche nach der Auffächerung der Kerngruppe der Brassicaceae in die Abstammungslinien stattgefunden haben muss.The glucosinolate-myrosinase system of the Brassicales is a plant defense system. Specifier proteins are part of this activated chemical defense system. Upon tissue disruption glucosinolates are hydrolysed by myrosinases. The arising hydrolysis products rearrange spontaneously to form toxic isothiocyanates which serve as defense compounds. In the presence of specifier proteins, epithionitriles, simple nitriles or organic thiocyanates are formed. The biological activity of the alternative products is largely unknown. The aim of this work was the cloning, characterisation and pyhlogenetic analyses of specifier proteins with different product specificities. Based on a phytochemical screening it was possible to detect hydrolyses products others than isothiocyanates in 14áspecies of the Brassicaceae and in one species of the Tropaeolaceae. Additional to the eight already known specifier proteins it was possible to isolate full-length cDNAs of three NSPs, six ESPs and two TFPs and to biochemically characterize the corresponding proteins.For the phylogenetic analyses of the specifier proteins and with comprehension of three not characterized proteins identified in a database, it was possible to include 22 full-length cDNAs and the homologous sequence At3g07720 from A. thaliana. The phylogenetic trees were calculated based on nucleotide and amino acid sequences using different methods. The obtained basal topology was identical for all investigated phylogenetic trees and together with the estimated data for sequence divergences and sequence identities as a measurement for evolutionary distances as well as together with the structural features of the specifier proteins it was possible to confirm At3g07720 as a member of a group of ancestral proteins. Based on At3g07720 and homologs the NSPs of the AtNSP5-cluster have evolved. A split of these NSPs lead to the NSPs of the NSP1-cluster. A following duplication of NSPs of the AtNSP1-cluster caused the ESPs and TFPs of the ESP/TFP-cluster. The expansion of ESPs could be estimated to a point in time after the expansion of the core group of the Brassicaceae in lineage I and lineage II and the preliminary gene duplication could be estimated to date back to at least 35,6 million years ago. The further evolution yielded to a twice independent genesis of TFPs from ESPs which could be dated after the expansion of the core group of the family Brassicaceae in the two lineages I and II

Taste and flavour perceptions of glucosinolates, isothiocyanates, and related compounds

Brassicaceae plants are renowned for their taste, aroma and trigeminal characteristics; predominantly bitter taste, sulfurous aroma and pungency. Compounds responsible for these sensations include the glucosinolates (GSLs) and their hydrolysis products, particularly isothiocyanates (ITCs), but also sulfur‐containing volatile compounds. This article reviews the relative importance of taste and flavour perceptions resulting from such compounds; collating evidence from papers where findings are based on sensory analytical correlations, and those that have extracted specific compounds prior to sensory evaluation. Where specific GSLs impart bitterness and many ITCs impart pungency, this is clearly not true for all GSLs and ITCs. Designing crop improvement strategies for sensory traits based on total GSL content would be flawed, as it does not consider the relative differences in sensory characteristics of different GSLs and ITCs, nor contribution from other GSL hydrolysis products. In addition, some Brassicaceae plants are consumed raw, whilst others are cooked; this affects not only the hydrolysis of GSLs, but also the generation and release of sulfides. Therefore, in breeding new plant varieties it is prudent to consider the individual GSLs, the typical cooking conditions the plant is subjected to, enzyme stability, and resultant composition of both GSL hydrolysis products (including ITCs) and sulfides

Der Code of Conduct der Universität Klagenfurt : ein Beitrag zur Sicherung guter wissenschaftlicher Praxis?

Mathias KuchernigKlagenfurt, Alpen-Adria-Univ., Dipl.-Arb., 2013(VLID)240919

Der Code of Conduct der Universität Klagenfurt : ein Beitrag zur Sicherung guter wissenschaftlicher Praxis?

Mathias KuchernigKlagenfurt, Alpen-Adria-Univ., Dipl.-Arb., 201

Sexuelle Unlust – Wirkmechanismen des natürlichen Aphrodisiakums Damiana (Turnera diffusa)

ZusammenfassungSexuelle Unlust bei Frauen ist eine häufig auftretende und meist multifaktorielle Symptomatik in jedem Alter. Entsteht daraus ein individueller Leidensdruck, einhergehend mit einer schlechten Lebensqualität, dann leiden Frauen daran umfassender und möglicherweise schwerwiegender als Männer. Ihre sexuellen Probleme werden jedoch im klinischen Umfeld oft nicht erkannt und bleiben unbehandelt, denn nur 20–34 % der Betroffenen suchen aktiv Hilfe bei einem Arzt. Die Auslöser mangelnder Libido können vielfältig sein und neben stressigen Lebensphasen auch an bestehende Medikationen, den demografischen Hintergrund, Probleme in der Partnerschaft oder Umstellungen der Hormone z. B. in den Wechseljahren gekoppelt sein. Pharmakotherapeutisch stehen jedoch nur relativ wenige Optionen für die Behandlung zur Verfügung. Neben einer nur in der Off-Label-Anwendung möglichen Verabreichung von transdermalem Testosteron oder Dehydroepiandrosteron, der Überweisung an andere Fachgebiete oder einer interdisziplinären Zusammenarbeit kann ein möglicher Baustein der Behandlung in der Verwendung geeigneter Phytopharmaka liegen. Durch eine Gesamtwirkung der beobachteten Einzelwirkungen von Inhaltsstoffen der Pflanze Damiana (Turnera diffusa) aus der Familie der Safranmalven (Turneraceae) können daraus hergestellte Arzneimittel eine gute Behandlungsoption mit einem positiven Einfluss auf eine verminderte Libido darstellen.</jats:p

Evolution of specifier proteins in glucosinolate-containing plants

<p>Abstract</p> <p>Background</p> <p>The glucosinolate-myrosinase system is an activated chemical defense system found in plants of the Brassicales order. Glucosinolates are stored separately from their hydrolytic enzymes, the myrosinases, in plant tissues. Upon tissue damage, e.g. by herbivory, glucosinolates and myrosinases get mixed and glucosinolates are broken down to an array of biologically active compounds of which isothiocyanates are toxic to a wide range of organisms. Specifier proteins occur in some, but not all glucosinolate-containing plants and promote the formation of biologically active non-isothiocyanate products upon myrosinase-catalyzed glucosinolate breakdown.</p> <p>Results</p> <p>Based on a phytochemical screening among representatives of the Brassicales order, we selected candidate species for identification of specifier protein cDNAs. We identified ten specifier proteins from a range of species of the Brassicaceae and assigned each of them to one of the three specifier protein types (NSP, nitrile-specifier protein, ESP, epithiospecifier protein, TFP, thiocyanate-forming protein) after heterologous expression in <it>Escherichia coli</it>. Together with nine known specifier proteins and three putative specifier proteins found in databases, we subjected the newly identified specifier proteins to phylogenetic analyses. Specifier proteins formed three major clusters, named AtNSP5-cluster, AtNSP1-cluster, and ESP/TFP cluster. Within the ESP/TFP cluster, specifier proteins grouped according to the Brassicaceae lineage they were identified from. Non-synonymous vs. synonymous substitution rate ratios suggested purifying selection to act on specifier protein genes.</p> <p>Conclusions</p> <p>Among specifier proteins, NSPs represent the ancestral activity. The data support a monophyletic origin of ESPs from NSPs. The split between NSPs and ESPs/TFPs happened before the radiation of the core Brassicaceae. Future analyses have to show if TFP activity evolved from ESPs at least twice independently in different Brassicaceae lineages as suggested by the phylogeny. The ability to form non-isothiocyanate products by specifier protein activity may provide plants with a selective advantage. The evolution of specifier proteins in the Brassicaceae demonstrates the plasticity of secondary metabolism within an activated plant defense system.</p

Sustainable Phenolic Fractions as Basis for Furfuryl Alcohol-Based Co-Polymers and Their Use as Wood Adhesives

Furfuryl alcohol is a very interesting green molecule used in the production of biopolymers. In the present paper, the copolymerization in acid environment with natural, easily-available, phenolic derivatives is investigated. The processes of polymerization of the furfuryl alcohol with: (i) spent-liquor from the pulping industry and (ii) commercial tannin from acacia mimosa were investigated though viscometry and IR-spectroscopy. The curing kinetics of the formulations highlighted the importance of the amount of furfuryl alcohol and catalyst as well as the effect of temperature for both phenolic-furanic polymers. Evidence of covalent copolymerization has been observed through infrared spectrometry (FT-IR) combined with principal component analysis (PCA) and confirmed with additional solubility tests. These bio-based formulations were applied as adhesives for solid wood and particleboards with interesting results: at 180 °C, the spent-liquor furanic formulations allow wood bonding slightly with lower performance than PVA in dry conditions, while mixed formulations allow the gluing of particleboard with only satisfactory internal bonding tests