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This paper is devoted the biotechnology development of Artemisia dracunculus L. genetic transformation. We obtained the transgenic A. dracunculus \"hairy\" root culture using A. rhizogenes A4-mediated transformation. The conditions of tarragon's genetic transformation were optimized. It was shown that leaves of in vitro cultivated plants were the optimal type of explants. The transgenic root formation frequency was up to 20% in case of leaves usage. The time of explants cocultivation with Agrobacterium suspension was found to be an important factor of biotechnology which affects the frequency of transgenic root growth. Transgenic root lines differed in morphological features and growth rate. Specific mass increase varied from 17 to 32 times after 3 weeks cultivation on 1/2 Murashige-Skoog medium.

Artemisia absinthium L. plants are known as producers of substances with antioxidant properties. Among others, phenols and flavonoids are found in these plants. The synthesis of these bioactive compounds can be activated by genetic transformation. This process can be carried out even without the transfer of specific genes involved in the synthesis of flavonoids. Thus, \"hairy\" roots, obtained after Agrobacterium rhizogenes - mediated transformation, can produce a variety of valuable substances. The aim of this study was obtaining A. absinthium \"hairy\" roots with high phenolic content. Methods. \"Hairy\" roots were obtained by co-cultivation leaves with suspension of A. rhizogenes with pCB124 vector. The presence of transferred genes was confirmed by PCR. The reactions with AlCl3 and Folin-Ciocalteu reagent were used to determine the total flavonoids and phenols content. The antioxidant activity of extracts was evaluated by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Results. PCR analysis detected the presence of bacterial rol genes and the absence of pCB124 plasmid genes. Root lines differed in growth rate. \"Hairy\" roots were characterized by a higher phenolic content, particularly flavonoids (up to 4.784 ± 0.10 mg/g FW) compared to control (3.861±0.13 mg/g FW). Also, extracts from transgenic roots demonstrated higher antioxidant activity in the reaction with DPPH reagent (EC50 = 3.6 57 mg) when compared with extracts from control plants (EC50 = 6,716 mg). Conclusions. A. rhizogenes-mediated transformation of A. absinthium can be applied for obtaining transgenic root lines with increased phenolic content and higher antioxidant activity.

Fullerene C 60 as a representative of carbon nanocompounds is suggested to be promising agent for application in photodynamic therapy due to its unique physicochemical properties. The goal of this study was to estimate the accumulation of fullerene C 60 in leukemic cells and to investigate its phototoxic effect on parental and resistant to cisplatin leukemic cells. Stable homogeneous water colloid solution of pristine C 60 with average 50-nm diameter of nanoparticles was used in experiments. Fluorescent labeled C 60 was synthesized by covalent conjugation of C 60 with rhodamine B isothiocyanate. The results of confocal microscopy showed that leukemic Jurkat cells could effectively uptake fullerene C 60 from the medium. Light-emitting diode lamp (100 mW cm −2 , λ = 420–700 nm) was used for excitation of accumulated C 60 . A time-dependent decrease of viability was detected when leukemic Jurkat cells were exposed to combined treatment with C 60 and visible light. The cytotoxic effect of photoexcited C 60 was comparable with that induced by H 2 O 2 , as both agents caused 50% decrease of cell viability at 24 h at concentrations about 50 μM. Using immunoblot analysis, protein phosphotyrosine levels in cells were estimated. Combined action of C 60 and visible light was followed by decrease of cellular proteins phosphorylation on tyrosine residues though less intensive as compared with that induced by H 2 O 2 or protein tyrosine kinase inhibitor staurosporine. All tested agents reduced phosphorylation of 55, 70, and 90 kDa proteins while total suppression of 26 kDa protein phosphorylation was specific only for photoexcited C 60 . The cytotoxic effect of C 60 in combination with visible light irradiation was demonstrated also on leukemic L1210 cells both sensitive and resistant to cisplatin. It was shown that relative value of mitochondrial membrane potential measured with tetramethylrhodamine ethyl ester perchlorate (TMRE) probe was lower in resistant cells in comparison with sensitive cells and the drop of mitochondrial potential corresponded to further decrease of resistant cell viability after C 60 photoexcitation. The data obtained allow to suggest that C 60 -mediated photodynamic treatment is a candidate for restoration of drug-resistant leukemic cell sensitivity to induction of mitochondrial way of apoptosis.

Aim. This study is focused on a comprehensive overview of mechanisms and processes involved in the acquisition of cancer cell plasticity in a manner dependent on the adapter protein Ruk/CIN85 (in rodents, Ruk — regulator of ubiquitous kinase; in human CIN85 — Cbl-interacting protein of 85 kDa, encoded by SH3KBP1 gene).. Methods. Gene expression was evaluated using RT2-PCR and Western blotting, cell proliferation and survival were analyzed using MTT and/or dye exclusion assays, motility was assessed by scratch test and Transwell assay, enzyme activities were measured using spectrophotometric assays. In vivo metastasis were studies using experimental metastasis model. Conclusion. This study discloses various aspects of cancer cells plasticity, such as EMT, stemness, metabolic changes, ECM components, and drug resistance in dependence on adaptor protein Ruk/CIN85 expression level.

The purpose of this study was to test the hypothesis that Ruk/CIN85 overexpression/knockdown in melanoma cells may be involved in the regulation of EMT. Materials and methods. The mouse melanoma cell line B16-F10 and its sublines with up-/down-regulation of Ruk/CIN85 (generated early using lentiviral technology) were used as a model for research. Melanoma cells were cultured in the complete RPMI 1610 medium under standard conditions. Proliferative activity of the cells was estimated using the MTT-test, and cell migratory potential was studied by the wound-healing assay. The data obtained were analyzed with parametric Student`s t-test. Results were expressed as mean ± SEM and significance was set at P<0.05. Results and Discussion. Cutaneous melanoma genesis is a multi-step process initiated by the transformation of a normal melanocyte following an oncogenic insult. Due to the transcriptome and metabolome reprogramming in the course of EMT, transformed melanoma cells change their phenotype and acquire increased proliferative rate, cell motility, invasiveness, and metastatic potential. According to the data obtained, overexpression of Ruk/CIN85 in B16 mouse melanoma cells (subclones Up7 and Up21) led to an increase in their proliferative activity by 1,6 and 1.8 times, respectively, at 24th hour in comparison with control Mock cells . At the 48th hour, when the cells reached confluence, the cell viability of subclones did not differ from the control ones. No statistically significant changes in the proliferative activity of B16 cells with suppressed expression of the adaptor protein (subclone Down) were found. In accordance with previous data, B16 cells overexpressing Ruk/CIN85 were characterized by strongly increased motility rate (more than twofold for both Up7 and Up21 subclones compared to control Mock cells). At the same time, knockdown of Ruk/CIN85 in B16 cells resulted in a decrease in their migratory activity by about 30%. Conclusions. All findings obtained demonstrated that the malignancy traits of melanoma B16 cells are inversely modulated upon up- and down-changes in adaptor protein Ruk/CIN85 expression levels suggesting its possible role in the control of EMT.

Aim. This study aimed to investigate the changes in glucose metabolism in mouse 4T1 breast adenocarcinoma cells with different levels of Ruk/CIN85 expression. Methods. We used 4T1 cells with stable overexpression (subline RukUp) or knockdown (subline RukDown) of Ruk/CIN85, as well as corresponding vector control sublines Mock and Scr. Cells were cultured in the complete RPMI-1640 medium under standard conditions. mRNA expression levels were estimated by RT2-PCR, enzymes activities were measured by spectrophotometric and/or fluorometric assays. Results. Analysis of mRNA expression of glucose metabolism-related genes in RukUp and RukDown cells revealed that glycolysis genes are preferentially overexpressed in RukUp cells, and downregulated in RukDown cells. Thus, RukUp cells were characterized by significantly overexpressed Slc2a1, Gck, Aldoa, and Ldha, while in RukDown cells these genes were either down regulated or not changed. However, the expression of TCA (tricarboxylic acid) cycle enzyme Mdh2 increased dramatically (by 7,8 times) in RukDown cells. In detail, we observed statistically significant changes in the activity of all studied enzymes in RukUp cells (increase by 1,5-1,9 times for glycolysis enzymes and G6PD, and decrease by 1,33-1,69 times for TCA enzymes). However, in RukDown cells we did not find any significant changes in glycolysis enzymes activities, but activities of mitochondrial IDH3 and MDH2 were elevated by 1,65 and 1,59 times, respectively. Conclusions. The results obtained indicate that adaptor protein Ruk/CIN85 is involved in the metabolic reprogramming during breast cancer progression. High level of Ruk/CIN85 expression is associated with potentiation of the Warburg effect.

The results of electrochemical processing of the ZhS32-VI refractory alloy implemented in a galvanostatic mode in the nitric acid electrolyte are described. Experiments on the electrochemical dissolution of the same alloy in the galvanostatic mode using a nitric acid solution with a concentration of 100 g/L at various current densities are carried out. It is shown that its constituent components separate quantitatively: refractory metals such as niobium, tantalum, molybdenum, and tungsten concentrate in the anodic slime, while partially cobalt and rhenium, as well as most of the aluminum, chromium, and nickel, transfer into the electrolyte. The functional process diagram for processing of the ZhS32-VI alloy is proposed, in which the formation and separation of the main mass of nickel and cobalt are performed at the first stage with the formation of the metallic Ni–Co-containing sediment.

Fullerene C as a representative of carbon nanocompounds is suggested to be promising agent for application in photodynamic therapy due to its unique physicochemical properties. The goal of this study was to estimate the accumulation of fullerene C in leukemic cells and to investigate its phototoxic effect on parental and resistant to cisplatin leukemic cells. Stable homogeneous water colloid solution of pristine C with average 50-nm diameter of nanoparticles was used in experiments. Fluorescent labeled C was synthesized by covalent conjugation of C with rhodamine B isothiocyanate. The results of confocal microscopy showed that leukemic Jurkat cells could effectively uptake fullerene C from the medium. Light-emitting diode lamp (100 mW cm , λ = 420-700 nm) was used for excitation of accumulated C . A time-dependent decrease of viability was detected when leukemic Jurkat cells were exposed to combined treatment with C and visible light. The cytotoxic effect of photoexcited C was comparable with that induced by H O , as both agents caused 50% decrease of cell viability at 24 h at concentrations about 50 μM. Using immunoblot analysis, protein phosphotyrosine levels in cells were estimated. Combined action of C and visible light was followed by decrease of cellular proteins phosphorylation on tyrosine residues though less intensive as compared with that induced by H O or protein tyrosine kinase inhibitor staurosporine. All tested agents reduced phosphorylation of 55, 70, and 90 kDa proteins while total suppression of 26 kDa protein phosphorylation was specific only for photoexcited C .The cytotoxic effect of C in combination with visible light irradiation was demonstrated also on leukemic L1210 cells both sensitive and resistant to cisplatin. It was shown that relative value of mitochondrial membrane potential measured with tetramethylrhodamine ethyl ester perchlorate (TMRE) probe was lower in resistant cells in comparison with sensitive cells and the drop of mitochondrial potential corresponded to further decrease of resistant cell viability after C photoexcitation. The data obtained allow to suggest that C -mediated photodynamic treatment is a candidate for restoration of drug-resistant leukemic cell sensitivity to induction of mitochondrial way of apoptosis.

Fullerene C60 as a representative of carbon nanocompounds is suggested to be promising agent for application in photodynamic therapy due to its unique physicochemical properties. The goal of this study was to estimate the accumulation of fullerene C60 in leukemic cells and to investigate its phototoxic effect on parental and resistant to cisplatin leukemic cells. Stable homogeneous water colloid solution of pristine C60 with average 50-nm diameter of nanoparticles was used in experiments. Fluorescent labeled C60 was synthesized by covalent conjugation of C60 with rhodamine B isothiocyanate. The results of confocal microscopy showed that leukemic Jurkat cells could effectively uptake fullerene C60 from the medium. Light-emitting diode lamp (100 mW cm-2, [lambda]=420-700 nm) was used for excitation of accumulated C60. A time-dependent decrease of viability was detected when leukemic Jurkat cells were exposed to combined treatment with C60 and visible light. The cytotoxic effect of photoexcited C60 was comparable with that induced by H2O2, as both agents caused 50% decrease of cell viability at 24 h at concentrations about 50 [mu]M. Using immunoblot analysis, protein phosphotyrosine levels in cells were estimated. Combined action of C60 and visible light was followed by decrease of cellular proteins phosphorylation on tyrosine residues though less intensive as compared with that induced by H2O2 or protein tyrosine kinase inhibitor staurosporine. All tested agents reduced phosphorylation of 55, 70, and 90 kDa proteins while total suppression of 26 kDa protein phosphorylation was specific only for photoexcited C60. The cytotoxic effect of C60 in combination with visible light irradiation was demonstrated also on leukemic L1210 cells both sensitive and resistant to cisplatin. It was shown that relative value of mitochondrial membrane potential measured with tetramethylrhodamine ethyl ester perchlorate (TMRE) probe was lower in resistant cells in comparison with sensitive cells and the drop of mitochondrial potential corresponded to further decrease of resistant cell viability after C60 photoexcitation. The data obtained allow to suggest that C60-mediated photodynamic treatment is a candidate for restoration of drug-resistant leukemic cell sensitivity to induction of mitochondrial way of apoptosis.

It is known for many years that iron represses synthesis of riboflavin (RF) and most of RF-synthesizing enzymes in several yeast species, known as flavinogenic yeasts. However, the mechanism of such repression is not known. We have found that iron represses transcription of RIB1 and RIB7 genes coding for the first and the last enzymes of RF biosynthesis in the model flavinogenic organism Pichia guilliermondii. To decipher molecular mechanisms of iron-dependent repression, isolation and study of the regulatory mutants defective in corresponding regulation is desirable. However, no suitable methods for isolation of such mutants were previously available. We have produced a single-point transition mutation in the RIB1 gene. The corresponding rib1-86 mutant exhibits leaky phenotype and is unable to grow in iron-sufficient minimal medium without exogenous RF. However, it can grow in minimal iron-deficient medium without RF, or in iron-sufficient medium upon introduction of the previously-isolated regulatory mutation rib81, which leads to increase in RF production. Using the rib1-86 mutant as parental strain, a collection of mutants able to grow in iron-sufficient medium without exogenous RF has been isolated. The mutants appeared to be defective in regulation of RF biosynthesis and iron homeostasis and were divided into six new complementation groups. Study of one corresponding mutant, red6, showed derepression of RIB1 mRNA synthesis in iron-sufficient medium.