Purification and preliminary characterization of a xylanase from Thermomyces lanuginosus strain SS-8

Thermomyces lanuginosus SS-8 was isolated from soil samples that had been collected from near self-heating plant material and its extracellular cellulase-free xylanase purified approximately 160-fold using ion exchange chromatography and continuous elution electrophoresis. This xylanase was thermoactive (optimum temperature 60 °C) at pH 6.0 and had a molecular weight of 23.79 kDa as indicated by SDS-PAGE electrophoresis. The xylanase rapidly hydrolyzed xylan directly to xylose without the production of intermediary xylo-oligosaccharides within 15 min of incubation under optimum conditions. This trait of rapidly degrading xylan to xylose as a sole end-product could have biotechnological potential in degradation of agro-wastes for bioethanol manufacturing industry

Differential contributions of peripheral and central mechanisms to pain in a rodent model of osteoarthritis

The mechanisms underlying the transition from acute nociceptive pain to centrally maintained chronic pain are not clear. We have studied the contributions of the peripheral and central nervous systems during the development of osteoarthritis (OA) pain. Male Sprague-Dawley rats received unilateral intra-articular injections of monosodium iodoacetate (MIA 1mg) or saline, and weight bearing (WB) asymmetry and distal allodynia measured. Subgroups of rats received intra-articular injections of, QX-314 (membrane impermeable local anaesthetic)+capsaicin, QX-314, capsaicin or vehicle on days 7, 14 or 28 post-MIA and WB and PWT remeasured. On days 7&14 post-MIA, but not day 28, QX-314+capsaicin signfcantly attenuated changes in WB induced by MIA, illustrating a crucial role for TRPV1 expressing nociceptors in early OA pain. The role of top-down control of spinal excitability was investigated. The mu-opioid receptor agonist DAMGO was microinjected into the rostroventral medulla, to activate endogenous pain modulatory systems, in MIA and control rats and refex excitability measured using electromyography. DAMGO (3ng) had a signifcantly larger inhibitory effect in MIA treated rats than in controls. These data show distinct temporal contribtuions of TRPV1 expressing nociceptors and opioidergic pain control systems at later timepoints

Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death

Method for biosynthesis of specific isotope-marked secondary metabolites

Die Erfindung betrifft ein Verfahren zur Biosynthese von spezifisch isotopenmarkierten Sekundärmetaboliten. Zielsetzung der vorliegenden Erfindung war die Entwicklung eines Verfahrens zur spezifischen Markierung von mikrobiellen Sekundärmetaboliten mit stabilen oder radioaktiven Isotopen, mit dem unter stark reduziertem Einsatz markierter Vorstufen höchste Einbau- und Markierungsraten erzielt werden können. Erfindungsgemäß werden mittels gentechnischer Methoden im Genom von bakteriellen bzw. pilzlichen Sekundärmetabolitproduzenten Gene der Aminosäurebiosynthese ausgeschaltet, deren Endprodukte (Aminosäuren) als Donoren funktioneller Gruppen von Sekundärmetaboliten dienen bzw. teilweise oder vollständig in die Molekülstruktur von Sekundärmetaboliten integriert werden. Der Einsatz isotopenmarkierter Substanzen erfolgt dabei wesentlich gezielter als bei dem gegenwärtig eingesetzten Verfahren zur Totalmarkierung, welches auf einer Zugabe von markierten Aminosäuren in ein Kulturmedium beruht. Das erfindungsgemäße Verfahren kann zur gezielten Markierung einer Vielzahl von Naturstoffen, eingesetzt werden. Die besondere Bedeutung des Verfahrens liegt dabei in der Herstellung preiswerter, verlässlicher Referenzsubstanzen/Standards für die moderne, hochsensitive Analytik-Verfahren wie SIDA-MS/MS.The invention relates to a method for biosynthesis of specific isotope-marked secondary metabolites. The invention addresses the problem of developing a method for specific marking of microbial secondary metabolites having stable or radioactive isotopes, by means of which maximum installation and marking rates can be achieved via strongly reduced use of marked precursors. According to the invention, genes of amino acid biosynthesis in the genome of bacterial or fungal secondary metabolite producers are deactivated by means of genetic engineering methods, the final products (amino acids) of which serve as donors of functional groups of secondary metabolites and are partially or completely integrated in the molecular structure of secondary metabolites. The use of isotope-marked substances occurs substantially more specifically according to the invention than in the currently used method of total marking, which is based on an addition of marked amino acids to a culture medium. The method according to the invention can be used for specific marking of a plurality of natural substances. The particular significance of the method lies in the production of inexpensive, reliable reference substances/standards for modern, highly sensitive analytics methods such as SIDA-MS/MS.L'invention concerne un procédé de biosynthèse de métabolites secondaires marqués spécifiquement par des isotopes. L'invention vise à développer un procédé de marquage spécifique des métabolites secondaires microbiens par des isotopes stables ou radioactifs, qui permette d'atteindre des taux d'incorporation et de marquage les plus élevés possible tout en réduisant fortement le recours à des étapes de pré-marquage. Le procédé selon l'invention consiste à neutraliser au moyen de méthodes génétiques dans le génome de producteurs de métabolites secondaires bactériens ou fongiques les gènes de la biosynthèse des acides aminés, dont les produits finaux (acides aminés) servent de donneurs de groupes fonctionnels de métabolites secondaires ou sont en partie ou en totalité intégrés dans la structure moléculaire de métabolites secondaires. L'utilisation de substances marquées par des isotopes se fait alors sensiblement de manière plus ciblée que dans le cas du procédé de marquage total utilisé actuellement, qui repose sur l'addition d'acides aminés marqués dans un milieu de culture. Le procédé selon l'invention peut être utilisé pour le marquage ciblé d'une pluralité de substances naturelles. L'importance particulière du procédé réside dans la production de substances de référence/de normes économiques et fiables pour des procédés d'analyse modernes hautement sensibles, par exemple les procédés d'analyse SIDA-MS/MS