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Publikation

Krägeloh, T.; Cavalleri, J. M. V.; Ziegler, J.; Sander, J.; Terhardt, M.; Breves, G.; Cehak, A. Identification of hypoglycin A binding adsorbents as potential preventive measures in co-grazers of atypical myopathy affected horses Equine Vet J 50, 220-227, (2018) DOI: 10.1111/evj.12723

BackgroundIntestinal absorption of hypoglycin A (HGA) and its metabolism are considered major prerequisites for atypical myopathy (AM). The increasing incidence and the high mortality rate of AM urgently necessitate new therapeutic and/or preventative approaches.ObjectivesTo identify a substance for oral administration capable of binding HGA in the intestinal lumen and effectively reducing the intestinal absorption of the toxin.Study designExperimental in vitro study.MethodsSubstances commonly used in equine practice (activated charcoal composition, di‐tri‐octahedral smectite, mineral oil and activated charcoal) were tested for their binding capacity for HGA using an in vitro incubation method. The substance most effective in binding HGA was subsequently tested for its potential to reduce intestinal HGA absorption. Jejunal tissues of 6 horses were incubated in Ussing chambers to determine mucosal uptake, tissue accumulation, and serosal release of HGA in the presence and absence of the target substance. Potential intestinal metabolism in methylenecyclopropyl acetic acid (MCPA)‐conjugates was investigated by analysing their concentrations in samples from the Ussing chambers.ResultsActivated charcoal composition and activated charcoal were identified as potent HGA binding substances with dose and pH dependent binding capacity. There was no evidence of intestinal HGA metabolism.Main limitationsBinding capacity of adsorbents was tested in vitro using aqueous solutions, and in vivo factors such as transit time and composition of intestinal content, may affect adsorption capacity after oral administration.ConclusionsFor the first time, this study identifies substances capable of reducing HGA intestinal absorption. This might have major implications as a preventive measure in cograzers of AM affected horses but also in horses at an early stage of intoxication.
Publikation

Ziegler, J.; Abel S. Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization Amino Acids 46, 2799-2808, (2014) DOI: 10.1007/s00726-014-1837-5

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performedusing l-norvaline as standard. A limit of detection as low as 1 fmol/μl with a linear range of up to 125 pmol/μl could be obtained. Intraday and interday precisions were lower than10 % relative standard deviations for most of the amino acids. Quantification usingl-norvaline as internal standard gave very similar results compared to the quantificationusing deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).
Publikation

Kopycki, J.; Schmidt, J.; Abel, S.; Grubb, D. Chemoenzymatic synthesis of diverse thiohydroximates from gluconsinolate-utilizing enymes from <i> Helix pomatia</i> and <i> Caldicellulosiruptor saccharolyticus</i> Biotechnol. Lett 33, 1039-1046, (2011) DOI: 10.1007/s10529-011-0530-y

Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a β-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed.
Publikation

Abel, S.; Savchenko, T.; Levy, M. Genome-wide comparative analysis of the <em>IQD</em> gene families in <em>Arabidopsis thaliana</em> and Oryza sativa BMC Evolutionary Biology 5, 72 (1-25), (2005)

We identified and analyzed 33 and 29 IQD1-like genes in Arabidopsis thaliana and Oryza sativa, respectively. The encoded IQD proteins contain a plant-specific domain of 67 conserved amino acid residues, referred to as the IQ67 domain, which is characterized by a unique and repetitive arrangement of three different calmodulin recruitment motifs, known as the IQ, 1-5-10, and 1-8-14 motifs. We demonstrated calmodulin binding for IQD20, the smallest IQD protein in Arabidopsis, which consists of a C-terminal IQ67 domain and a short N-terminal extension. A striking feature of IQD proteins is the high isoelectric point (~10.3) and frequency of serine residues (~11%). We compared the Arabidopsis and rice IQD gene families in terms of gene structure, chromosome location, predicted protein properties and motifs, phylogenetic relationships, and evolutionary history. The existence of an IQD-like gene in bryophytes suggests that IQD proteins are an ancient family of calmodulin-binding proteins and arose during the early evolution of land plants. Comparative phylogenetic analyses indicate that the major IQD gene lineages originated before the monocot-eudicot divergence. The extant IQD loci in Arabidopsis primarily resulted from segmental duplication and reflect preferential retention of paralogous genes, which is characteristic for proteins with regulatory functions. Interaction of IQD1 and IQD20 with calmodulin and the presence of predicted calmodulin binding sites in all IQD family members suggest that IQD proteins are a new class of calmodulin targets. The basic isoelectric point of IQD proteins and their frequently predicted nuclear localization suggest that IQD proteins link calcium signaling pathways to the regulation of gene expression. Our comparative genomics analysis of IQD genes and encoded proteins in two model plant species provides the first step towards the functional dissection of this emerging family of putative calmodulin targets.
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