2,472 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
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
Energy conservation in modified Nyström methods for separable Hamiltonian systems
No full textNumerical integration methods that preserve structural properties of time dependent differential equations have often improved stability and smaller errors in long-term simulations than classical standard methods like Runge-Kutta or BDF. In the present paper an energy conserving Galerkin type approach of Betsch and Steinmann is generalized to modified Nyström methods for separable Hamiltonian systems. The benefits of this new class of methods are illustrated by numerical tests for a benchmark problem from celestial mechanics
Dahlquist's classical papers on stability theory
No full textThis text, which is based on the author's talk in honour of G. Dahlquist at the SciCade05 meeting in Nagoya, describes the two classical papers from 1956 and 1963 of Dahlquist and their enormous impact on the research of decades to come; it also allows the author to present a personal testimony of his never ending admiration for the scientific and personal qualities of this great ma
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