Acetaminophen overdose is the most frequent cause of acute liver injury. The main mechanism of acetaminophen toxicity has been attributed to oxidation of acetaminophen. The oxidation product is very reactive and reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). Although this conjugate has been recognized to be generally nontoxic, we have found recently that APAP-SG could produce a toxic effect. Therefore, the aim of our study was to estimate the toxicity of purified APAP-SG by characterizing the inhibitory effect in human glutathione reductase (GR) and comparing that to the inhibitory effect of the natural inhibitor reduced glutathione. We used two types of human GR: recombinant and freshly purified from red blood cells. Our results show that GR was significantly inhibited in the presence of both APAP-SG and reduced glutathione. For example, the enzyme activity of recombinant and purified GR was reduced in the presence of 4 mm APAP-SG (with 0.5 mm glutathione disulfide) by 28% and 22%, respectively. The type of enzyme inhibition was observed to be competitive in the cases of both APAP-SG and glutathione. As glutathione inhibits GR activity in cells under physiological conditions, the rate of enzyme inhibition ought to be weaker in the case of glutathione depletion that is typical of acetaminophen overdose. Notably, however, enzyme activity likely remains inhibited due to the presence of APAP-SG, which might enhance the pro-oxidative status in the cell. We conclude that our finding could reflect some other pathological mechanism that may contribute to the toxicity of acetaminophen.

• MeSH
• erytrocyty enzymologie   MeSH
• glutathion toxicita   MeSH
• glutathiondisulfid metabolismus   MeSH
• glutathionreduktasa antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• paracetamol analogy a deriváty   toxicita   MeSH
• rekombinantní proteiny metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakty MeSH

Specific allergen immunotherapy is frequently associated with adverse reactions. Several strategies are being developed to reduce the allergenicity while maintaining the therapeutic benefits. Peptide immunotherapy is one such approach. Methods for the simple and rapid identification of immunogenic epitopes of allergens (i.e. allergenic epitopes) are ongoing and could potentially lead to peptide-based vaccines. An epitope extraction technique, based on biofunctionalized magnetic microspheres self-organized under a magnetic field in a channel of a simple microfluidic device fabricated from polydimethylsiloxane, was applied in the isolation and identification of prospective allergenic epitopes. Similarly to chromatographic column separations, the easily replaceable plug of self-organized beads in the channel benefits especially from an even larger surface-to-volume ratio and an enhanced interaction of the surfaces with passing samples. Ovalbumin, the major protein of egg white and a typical representative of food allergens, was selected as the model molecule. Highly resistant ovalbumin was at first efficiently digested by a magnetic proteolytic reactor with trypsin treated with l-1-tosylamido-2-phenylethyl chloromethyl ketone and the second step, i.e. capture of allergenic epitopes from the mixture of peptides, was performed by a magnetic immunoaffinity carrier with orientedly immobilized rabbit anti-ovalbumin IgG molecules. Captured peptides were released with 0.05% trifluoroacetic acid. The elution fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The peptide fragment of ovalbumin HIATNAVLFFGR (m/z: 1345.75, position: 371-382) was identified as a relevant allergenic epitope in this way. Such a microfluidic magnetic force-based epitope extraction technique applied in the epitope mapping of ovalbumin has the potential to be a significant step towards developing safe and cost-effective epitope-based vaccines.

• MeSH
• alergeny chemie   imunologie   MeSH
• epitopy analýza   MeSH
• financování organizované MeSH
• hmotnostní spektrometrie MeSH
• imunomagnetická separace metody   MeSH
• mapování epitopu metody   MeSH
• mikrofluidní analytické techniky metody   MeSH
• mikrosféry MeSH
• ovalbumin chemie   imunologie   MeSH
• potravinová alergie MeSH
• vakcíny MeSH

We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.

• MeSH
• bioreaktory MeSH
• časové faktory MeSH
• chromatografie afinitní metody   přístrojové vybavení   MeSH
• enzymy imobilizované chemie   MeSH
• financování organizované MeSH
• lidé MeSH
• ligandy MeSH
• magnetismus MeSH
• metaloendopeptidasy chemie   MeSH
• nanočástice chemie   MeSH
• neurotensin analýza   MeSH
• oxid křemičitý MeSH
• peptidy analýza   chemie   MeSH
• proteomika MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• serinové endopeptidasy chemie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• trypsin chemie   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH