Tag Archives: MTT test
Honeybee chitosan-melanin complex: isolation and investigation of antimicrobial activity
M. Lootsik1, N. Manko1, O. Gromyko2,
S. Tistechok2, M. Lutsyk (Jr.)3, R. Stoika1*
1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv;
2Ivan Franko National University of Lviv, Ukraine;
3Danylo Halytsky Lviv National Medical University, Ukraine;
*e-mail: stoika.rostyslav@gmail.com
Received: 4 May 2020; Accepted: 13 November 2020
Antimicrobial activity of marine crustaceans chitosans is well studied and is widely used in medicine, while chitosans of insects are poorly investigated in this aspect, though they might also be of practical significance. The aim of this study was to isolate and purify chitosan-melanin complex (CMC) from the honeybee corpses and to estimate its antimicrobial activity. Antibacterial activity of CMC was evaluated by MTT test, antifungal activity towards Candida albicans was estimated by calculating colony forming units (CFU method). The modified method of CMC isolation and purification was described which differs from the known analogs in deacetylation of chitin-melanin complex by its hydrolysis in 40% NaOH without previous melanin elimination and in further purification of CMC by differential solubilization at distinct pH values. The anti-microbial activity of CMC was characterized by prevalence of candidacidal effect, IC50 towards laboratory strain of C. albicans was 50 μg/ml. The ranking of studied bacteria sensitivity to the CMC action decreased as: E. coli > St. aureus > Ps. aeruginosa. It is suggested that CMC isolated from the honeybee corpses might be a perspective constituent of medicinal compositions for treatment of lesions caused by C. albicans infection.
Plasminogen modulates formation of reactive oxygen species in human platelets
A. A. Tykhomyrov, D. D. Zhernosekov, M. M. Guzyk, V. V. Korsa, T. V. Grinenko
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: artem_tykhomyrov@ukr.net
Reactive oxygen species (ROS) are considered to be important signalling molecules controlling many platelet functions. ROS production has been shown to be augmented by platelet activation, however, plasminogen (Pg) has not been studied in the context of modulating intraplatelet ROS levels. The aim of this study was to investigate the ability of different Pg forms to affect platelet metabolic activity/survival and intracellular ROS production in resting and activated platelets. Platelets isolated from donor plasma were pre-treated with Glu- or Lys-Pg (1.2 µM) and activated by thrombin (1.0 NIH unit/ml) or collagen (1.25 mg/ml). MTT assay was adapted to estimate total mitochondrial dehydrogenase activity, while intracellular ROS levels were monitored with the use of H2DCF-DA probe by flow cytometry. Lys-Pg was shown to slightly, but significantly, mitigate MTT reduction (P < 0.05 vs. control platelets). Two-fold elevation in metabolic activity of platelets stimulated by thrombin as compared to untreated cells was observed. However, this activation was less exhibited in the case of platelets pre-incubated with either Glu- of Lys-Pg, with a predominant effect of Lys-Pg. Unlike thrombin, collagen treatment dramatically suppressed metabolic activity of platelets by 60% compared to control (P < 0.05). Glu- or Lys-Pg pre-incubation had no effects on the activity of collagen-stimulated platelets. Two subpopulations of platelets were observed with distinct characteristics of intracellular ROS formation. Elevated ROS production was demonstrated in these populations of both thrombin- and collagen-treated platelets. Pg (Lys-form to greater extent) enhanced intracellular ROS generation in thrombin-stimulated platelets. These findings suggest that augmented ROS generation within platelets pre-treated with Pg followed by their stimulation may result in down-regulation of their survival and functional activity. This study adds to our understanding one more possible mechanism of Pg impact on the platelet function.