Tyrosinases act in the development of organoleptic properties of tea, raisins, etc., but also cause unwanted browning of fruits, vegetables, and mushrooms. The tyrosinase from Agaricus bisporus has been used as a model to study tyrosinase inhibitors, which are also indispensable in the treatment of skin pigmentation disorders. However, this model has disadvantages such as side enzyme activities and the presence of multiple isoenzymes. Therefore, we aimed to introduce a new tyrosinase model. The pro-tyrosinase from Polyporus arcularius was overproduced in Escherichia coli. Trypsin digestion led to a cleavage after R388 and hence enzyme activation. The tyrosinase was a homodimer and transformed L-DOPA and tert-butylcatechol preferentially. Various aurons were examined as effectors of this enzyme. 2'- and 3'-hydroxyaurones acted as its activators and 2',4'-dihydroxyaurone as an inhibitor, whereas 4'-hydroxyaurones were its substrates. The enzyme is a promising model for tyrosinase effector studies, being a single isoenzyme and void of side enzyme activities.
- Klíčová slova
- Escherichia coli, Polyporus arcularius, aurones, trypsin digestion, tyrosinase,
- MeSH
- benzofurany metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- kinetika MeSH
- Polyporus enzymologie MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- tyrosinasa chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aurone MeSH Prohlížeč
- benzofurany MeSH
- fungální proteiny MeSH
- rekombinantní proteiny MeSH
- tyrosinasa MeSH
Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. γH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, γH2AX foci and their co-localization induced by γ-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of γH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses ≥ 5 cGy when analyzed by IFC. Analysis of the co-localizing foci by ImageStream and Metafer showed statistical significance at doses ≥ 2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of γH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating γH2AX foci positive (with 1 or more γH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ≥ 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF.
- Klíčová slova
- 53BP1, ImageStream, Metafer, human lymphocytes, imaging flow cytometry, ionizing radiation, γH2AX,
- MeSH
- 53BP1 metabolismus MeSH
- fluorescenční mikroskopie MeSH
- histony metabolismus MeSH
- lidé MeSH
- lymfocyty metabolismus účinky záření MeSH
- poškození DNA * MeSH
- průtoková cytometrie metody MeSH
- software MeSH
- vztah dávky záření a odpovědi MeSH
- záření gama * MeSH
- zobrazování trojrozměrné metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 53BP1 MeSH
- H2AX protein, human MeSH Prohlížeč
- histony MeSH
- TP53BP1 protein, human MeSH Prohlížeč
