Background Dual anti-platelet therapy consisting of aspirin (ASA) and a P2Y12 inhibitor is a standard treatment to prevent thrombotic events in patients with acute coronary syndrome or myocardial ...infarction.
Objective
β‐Blockers require careful initiation and titration when used in patients with heart failure. Some patients tolerate β‐blocker therapy initiation without difficulty, whereas in other ...patients this period presents clinical challenges. We tested the hypothesis that polymorphisms at codons 389 (Arg389Gly) and 49 (Ser49Gly) of the β1‐adrenergic receptor would be associated with differences in initial tolerability of β‐blocker therapy in patients with heart failure. We also tested whether polymorphisms in the β2‐adrenergic receptor, G‐protein αs subunit (Gsα), and cytochrome P450 (CYP) 2D6 genes or S‐metoprolol plasma concentrations were associated with β‐blocker tolerability.
Methods
Sixty‐one β‐blocker‐naive patients with systolic heart failure were prospectively enrolled. Patients began taking 12.5 to 25 mg metoprolol controlled release/extended release with titration every 2 weeks (as tolerated) to 200 mg/d or the maximum tolerated dose over a period of 8 to 10 weeks. Decompensation was the composite of death, heart failure hospitalization, increase in other heart failure medications, or need to discontinue metoprolol. End points were assessed during the titration period.
Results
The overall rate of decompensation was not different between the codon 49 or 389 genotypes. However, a significantly greater percentage of patients with the Gly389 variant required increases in heart failure medications as compared with Arg389 homozygotes (48% versus 14%, respectively; P = .006). Similarly, patients with the Ser49 homozygous genotype were significantly more likely to require increases in concomitant heart failure therapy as compared with Gly49 carriers (41% versus 11%, respectively; P = .03). Neither CYP2D6 genotypes nor metoprolol pharmacokinetics differed between patients with and those without a decompensation event. There was no association between the β2‐adrenergic receptor or Gsα polymorphisms with decompensated heart failure.
Conclusions
Patients with the Gly389 variant and Ser49Ser genotype were significantly more likely to require increases in heart failure medications during β‐blocker titration and thus may require more frequent follow‐up during titration.
Clinical Pharmacology & Therapeutics (2005) 77, 127–137; doi: 10.1016/j.clpt.2004.10.006
We report a general method for screening, in solution, the impact of deviations from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how ...fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed "reference" duplex (usually a Watson-Crick duplex) and a related "test" duplex containing a lesion/alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a fluorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized fluorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.
SNP genotyping typically incorporates a review step to ensure that the genotype calls for a particular SNP are correct. For high-throughput genotyping, such as that provided by the GenomeLab ...SNPstream instrument from Beckman Coulter, Inc., the manual review used for low-volume genotyping becomes a major bottleneck. The work reported here describes the application of a neural network to automate the review of results.
We describe an approach to reviewing the quality of primer extension 2-color fluorescent reactions by clustering optical signals obtained from multiple samples and a single reaction set-up. The method evaluates the quality of the signal clusters from the genotyping results. We developed 64 scores to measure the geometry and position of the signal clusters. The expected signal distribution was represented by a distribution of a 64-component parametric vector obtained by training the two-layer neural network onto a set of 10,968 manually reviewed 2D plots containing the signal clusters.
The neural network approach described in this paper may be used with results from the GenomeLab SNPstream instrument for high-throughput SNP genotyping. The overall correlation with manual revision was 0.844. The approach can be applied to a quality review of results from other high-throughput fluorescent-based biochemical assays in a high-throughput mode.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
High-efficiency prefractionation of complex protein mixtures is critical for top-down proteomics, i.e., the analysis of intact proteins by MS. Free-flow electrophoresis (FFE) can be used for IEF to ...separate proteins within a pH gradient according to their pIs. In an FFE system, this separation is performed entirely in the liquid phase, without the need for particulate chromatographic media, gels, or membranes. Herein, we demonstrated the compatibility of IEF-FFE with ESI-Fourier transform ICR MS (ESI-FTICR-MS) for top-down experiments. We demonstrated that IEF-FFE of intact proteins were highly reproducible between FFE instruments, between laboratories, and between analyses. Applying native (0.2% hydroxypropylmethyl cellulose) IEF-FFE to an enzyme resulted in no decrease in enzyme activity; applying either native or denaturing (8 M urea) IEF-FFE to a four-protein mixture with different pIs resulted in isolation of each protein into separate fractions in a 96-well plate. After desalting, each protein was sequenced by top-down MS/MS. As an application of this technique, chicken erythrocyte histone H2A-IV and its major modified forms were enriched by IEF-FFE. Top-down analysis revealed Lys-5 to be a major acetylation site, in addition to N-terminal acetylation.
In the current study, we have developed a method to measure relative peptide stability over time in different blood collection tubes. Reversed-phase chromatography and liquid ...chromatography-matrix-assisted laser desorption/ionization were performed on three subjects to facilitate a deeper look into the plasma peptidome. The data further support the importance of protease inhibitors in stabilizing plasma samples. Significantly, we have revealed subject-to-subject variability in the intrinsic damage over time that is possible in standard EDTA plasma, with such variations extensively minimized by protease inhibitors. We conclude that protease inhibitors can simultaneously improve time-dependent and individual-dependent preanalytical variables in human plasma samples.
Mass spectrometry is the tool of choice for sequencing peptides and determining the sites of posttrans-lational modifications; however, this bottom-up approach lacks in providing global information ...about the modification states of proteins including the number and types of isoforms and their stoichiometry. Recently, various techniques and mass spectrometers, such as high-field Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers, have been developed to study intact proteins (top-down proteomics). While the protein molecular mass and the qualitative and quantitative information about protein isoforms can be revealed by FTICR-MS analysis, their primary structure (including the identification of modifications and their exact locations in the amino acid sequence) can directly be determined using the MS/MS capability offered by the FTICR mass spectrometer. The distinct advantage of top-down methods are that modifications can be determined for a specific protein isoform rather than for peptides belonging to one or several isoforms. In this chapter, we describe different top-down proteomic approaches enabled by high-field (7, 9.4, and 12 T) FTICR mass spectrometers, and their applicability to answer biological and biomedical questions. We also describe the use of the free flow electrophoresis (FFE) to separate proteins prior to top-down mass spectrometric characterization.
Adiponectin is an important adipocytokine hormone which circulates in blood as homo-oligomers (trimer, hexamer and high molecular weight (HMW) forms) as well as a truncated form corresponding to the ...globular domain. Free flow electrophoresis (FFE) used in zone electrophoresis mode revealed the presence of isoforms within these oligomeric forms in plasma. HMW adiponectin oligomer showed two isoforms which carry different charge density at pH 4.7, only one of which is susceptible to dissociation by SDS. The adiponectin hexamer was shown to consist of a doublet and also shown to have at least two isoforms. A truncated form of adiponectin was identified as the main constituent of adiponectin in plasma and appeared to circulate bound to a basic protein, potentially one of the chemokines reported to bind to the globular domain. Analysis of the monomer composition of the oligomers revealed differences in monomeric isoforms used to build up the oligomers.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Blood samples collected for proteome studies are subject to a variety of preanalytical instability, among which intrinsic proteolysis activities cause a broad spectrum of protein and peptide ...degradation. This chapter describes two MALDI MS-based methods for plasma peptidomic analyses; a direct MALDI-TOF MS and an LC MALDI-TOF MS. Using these methods, we compared peptides and their time-dependent changes in traditional serum, four plasma samples with different anticoagulants and additives: EDTA-based, citrate-based, or heparin-based, and EDTA-based with protease inhibitors. For minimizing plasma sample instability and preanalytical variation, we suggest using an optimized blood collection device, minimizing the dwell time during blood collection and handling, controlling centrifugation and handling at room temperature, and saving plasma samples for use at most one freeze/thaw cycle. We have optimized our protocol to achieve reproducibility in peptidomic analyses of plasma samples using MALDI-TOF MS by minimizing preanalytical and analytical variability.
Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a ...six-ring hairpin polyamide, ImPyPy-γ -PyPyPy-β -Dp (where Im = imidazole, Py = pyr-role, γ = γ -aminobutyric acid, β = β -alanine, and Dp = dimethylaminopropylamide), reveals an ≈ 1-2 kcal/mol greater affinity for the designated match site, 5′-TGTTA-3′, relative to the single base pair mismatch sites, 5′-TGGTA-3′ and 5′-TATTA-3′. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by Im-PyPy-γ -PyPyPy-β -Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.
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