The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1-10 μM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10-5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).

• MeSH
• antibakteriální látky toxicita   MeSH
• antimycin A analogy a deriváty   toxicita   MeSH
• azidy toxicita   MeSH
• buněčné dýchání účinky léků   MeSH
• chloramfenikol toxicita   MeSH
• citrátový cyklus účinky léků   MeSH
• deoxyglukosa toxicita   MeSH
• fluoracetáty toxicita   MeSH
• glykolýza účinky léků   MeSH
• malonáty toxicita   MeSH
• proteosyntéza účinky léků   MeSH
• pyruváty toxicita   MeSH
• reprodukovatelnost výsledků MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• stav beztíže MeSH
• streptomycin toxicita   MeSH
• Synechococcus chemie   účinky léků   izolace a purifikace   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40μM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples.

• MeSH
• biotest ekonomika   metody   MeSH
• buněčné linie MeSH
• cisplatina toxicita   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie ekonomika   metody   MeSH
• glutathion analýza   metabolismus   MeSH
• lidé MeSH
• malonáty toxicita   MeSH
• o-ftalaldehyd chemie   MeSH
• průtoková cytometrie MeSH
• pyrazoly chemie   MeSH
• senzitivita a specificita MeSH
• studie proveditelnosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH