104,179 research outputs found
Order stars and stiff integrators
No full textAbstractOrder stars, introduced in G. Wanner, E. Hairer, S.P. Nørsett (Order stars and stability theorems, BIT 18 (1978) 475–489), have become a fundamental tool for the understanding of order and stability properties of numerical methods for stiff differential equations. This survey retraces their discovery and their principal achievements. We also sketch some later extensions and describe some recent developments
Substituted nucleosides as potential inhibitors of purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP)
No full textPurine nucleoside phosphorylase (PNP, EC 2.4.2.1) from Mycobacterium tuberculosis (MtPNP) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. Although MtPNP and the human enzyme (HsPNP) belong to the same family, slight differences in transition states[1] and structural features[2] may be exploited to achieve specific inhibition.
To this aim seven 8-halo-, 8-alkylamino- and 8-alkoxypurine ribonucleosides (Figure 1) were synthesized by either direct regioselective halogenation of the corresponding substrate or halogen substitution with a nucleophile on the 8-bromo intermediate. All products were prepared in very good yield (58-89%) and isolated in high purity.
These nucleoside analogues were then submitted to a new 96-well LC-ESI-MS/MS method to assess their inhibition activity towards MtPNP and HsPNP, by monitoring the phosphorolysis of inosine to hypoxanthine. The enzymatic assay was performed with an overall time of about 15 min/plate for sample processing and 2 min/sample for LC-MS analysis. Validation of the quantification method met FDA criteria. Preliminary experimental data showed that the activity exerted towards MtPNP was negligible in most cases, with only 1 and 3 resulting to be weak inhibitors (at a μM scale). Although the inhibition results were not remarkable and further investigations are currently in progress, a safe and rapid screening assay towards MtPNP and HsPNP was developed.
References:
[1] Lewandowicz, A. et al. Biochemistry 2003, 42, 6057-6066.
[2] a) Caceres, R. A. et al. Biochimie 2012, 94, 155-165; b) Ducati, R. G. et al. Bioorg. Med. Chem. 2010, 18, 4769-4774
Activity assay of Purine Nucleoside Phosphorylases by LC-MS/MS
No full textIn the search for new therapeutics, fast and automated screening tools of chemical libraries are required for hit selection.
Nucleoside phosphorylases (NPs, E.C. 2.4.2) are among the key enzymes in nucleotide salvage/recycling pathway. NPs catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate (Pi) to generate the nucleobase and α-D-(deoxy)ribose-1-phosphate (see Scheme 1).[1]
NPs are also essential for the metabolism of nucleotides in bacteria and other organisms. Nucleotide metabolic pathways in lower organisms represent reasonable targets for chemotherapy as they usually differ from the human counterparts.1b Inhibition of pathogen purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) might result in the impairment of replicative processes, thus providing a new potential route to infection control.[2]
Here we describe a novel LC-MS/MS method for the assessment of the activity of PNPs as an alternative to routinely used assays.1b Enzymatic activity was assessed by phosphorolysis of inosine to hypoxanthine (Scheme 1). Kinetic parameters (Km, Vmax, Kcat) were determined with respect to inosine and Pi.
The assay was performed in a 96 well plate format with an overall reaction time of about 15 minutes per plate, followed by the application of HILIC-LC-MS/MS method for the rapid quantification of the produced hypoxanthine (less than 2 minutes for sample).
For method development and validation, a PNP from Aeromonas hydrophila was used due to accumulated data on this enzyme by our team over the years.[3]
The newly developed LC-MS/MS assay will be applied to the screening of potential inhibitors against pathogenic PNPs.
[1] a. Pugmire, M. J. et al. Biochem. J. 2002, 361, 1; b. Bzowska, A. et al. Pharmacol. Ther. 2000, 88, 349.
[2] a. de Moraes, M. C. et al. Anal. Bioanal. Chem. 2013, 405, 4871; b. Ducati, R. G. et al. Curr. Med. Chem. 2011, 18, 1258; c. Madrid, D. C. et al J. Biol. Chem. 2008, 283, 35899.
[3] a. Ubiali, D. et al. Adv. Synth. Catal. 2012, 354, 96; b. Serra, I. et al. ChemPlusChem 2013, 78, 157; c. Calleri, E. et al. J. Chromatogr. B 2014, 968, 79
Simultaneous Multiple MS Binding Assays for the Dopamine, Norepinephrine, and Serotonin Transporters
No full textIn this work, we present label-free, mass-spectrometry-based binding assays (MS Binding Assays), targeting the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT) in simultaneous binding experiments. Using a validated LC-ESI-MS/MS method for quantification of the selective dopamine transporter inhibitor (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol ((R,R)-D-84), the selective norepinephrine transporter inhibitor (S,S)-reboxetine, and the selective serotonin reuptake inhibitor (S)-citalopram, binding affinities at the three monoamine transporters could be characterized simultaneously in a single binding experiment. The performed simultaneous saturation and competition experiments yielded results that are in good accordance with those determined in MS Binding Assays addressing the monoamine transporters individually. The results obtained from this study underscore the potential of MS Binding Assays for simultaneous affinity determination at different targets, which is difficult to accomplish with conventional radioligand binding assays
Bibliographie Hilarion G. Petzold 1958 – 2009 mit Anhang als Einführung
No full textDieses Archiv enthält die Gesamtbibliographie der Werke des Autors nebst einiger Texte „Über H. G. Petzold“ im Schlussteil der Bibliographie sowie einen Anhang mit einer Einführung in die Architektur des Werkes in seinem wissenslogischen Aufbau als Ausarbeitung seines „Tree of Science Modells“ (2007).This archive contains the complete bibliography of the author and some texts about H. G. Petzold, moreover an epilogue with an introduction to the architecture of the works in its epistemological structure and composition and as an elaborations of Petzold’s „Tree of Science Modell (2007).https://www.fpi-publikation.de/polyloge/01-2009-petzold-h-g-gesamtbibliographie-h-g-petzold-1958-2009-updating-november2009/peerReviewedpublishedVersio
Dispelling the Myths Behind First-author Citation Counts
No full textWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Chromosome Centromeres: Structural and Analytical Investigations with High Resolution Scanning Electron Microscopy in Combination with Focused Ion Beam Milling
No full textWhole mount mitotic metaphase chromosomes of different plants and animals were investigated with high resolution field emission scanning electron microscopy (FESEM) to study the ultrastructural organization of centromeres, including metacentric, acrocentric, telocentric, and holocentric chromosome variants. It could be shown that, in general, primary constrictions have distinctive ultrastructural features characterized by parallel matrix fibrils and fewer smaller chromomeres. Exposure of these structures depends on cell cycle synchronization prior to chromosome isolation, chromosome size, and chromosome isolation technique. Chromosomes without primary constrictions, small chromosomes, and holocentric chromosomes do not exhibit distinct ultrastructural elements that could be directly correlated to centromere function. Putative spindle structures, although rarely observed, spread over the primary constriction to the bordering pericentric regions. Analytical FESEM techniques, including specific DNA staining with Pt blue, staining of protein as a substance class with silver-colloid, and artificial loosening of fixed chromosomes with proteinase K, were applied, showing that centromere variants and ultrastructural elements in the centromere differ in DNA and protein distribution. Immunogold localization allowed high-resolution comparison between chromosomes with different centromere orientations of the distribution of centromere-related histone variants, phosphorylated histone H3 (ser10), and CENH3. A novel application of FESEM combined with focused ion beam milling (FIB) provided new insights into the spatial distribution of these histone variants in barley chromosomes. Copyright (C) 2009 S. Karger AG, Base
- …
