Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF(103) of the isolate Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.
Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The K D values of galactose and meta-nitrophenyl α-D-galactoside were determined with weak affinity chromatography to be 52 and 1 mM, respectively, which agree well with IC 50 values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but K D values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct K D values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis.Keywords affinity constant; carbohydrate interactions; cholera toxin; high-performance liquid chromatography; receptor; ligand interactions; weak affinity chromatography Carbohydrate structures on mammalian epithelial cells are targeted by micro-organisms for specific attachment and initiation of infection or toxic effect. A specific inhibition of this initial interaction is an attractive strategy to control viral and bacterial infections (1). Anti-adhesion drugs could, for example, be of outmost importance in defeating cholera and traveller's diarrhea (2). The causative agents in these diseases are the toxins produced by Vibrio cholerae and Escherichia coli, respectively. The two toxins, cholera toxin (CT) and the heat-labile enterotoxin (LT), have a homology of about 80% and are essentially identical in binding and toxic effect, even though LT causes less severe symptoms than CT (3). They belong to the *Corresponding author: Maria Bergström, E-mail: maria.bergstrom@hik.se. NIH Public Access Author ManuscriptChem Biol Drug Des. Author manuscript; available in PMC 2010 January 1. Published in final edited form as:Chem Biol Drug Des. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript heterohexameric AB 5 bacterial toxins which all have an organization comprising a toxic Asubunit and an independent homopentamer of B-subunits, responsible for the specific recognition of glycosphingolipids on epithelial cells. The natural target in the case of CT and LT is the ganglioside GM1 (Galβ1-3GalNAcβ1-4[NeuAcα2-3]Galβ1-4Gl...
The article analyses what the third EU Directive on AML (anti-money laundering) and risk management means in terms of democratic accountability when the banking sector is given a role that is traditionally the prerogative of the public actors. The comparison between the UK and Sweden on the private actors' role in various stages of the risk-based decision process shows that the procedures used could jeopardize the traditional liberal understanding of democratic accountability.
A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using alpha-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors.
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