In the present work, we investigated the dark and photoinduced cytotoxic activity of the new chlorophyll-a derivatives which contain the substituents of oligoethylene glycol on the periphery of their macrocycles. These compounds were tested using human cell lines to estimate their potential as photosensitizers for photodynamic therapy of cancer. It was shown that all the tested compounds have expressed photoinduced cytotoxic activity in vitro. Detailed study of the biological activity of one of the most perspective compound in this series—pyropheophorbide-a 17-diethylene glycol ester (Compound 21) was performed. This new compound is characterized by lower dark cytotoxicity and higher photoinduced cytotoxicity than previously described in a similar compound (DH-I-180-3) and clinically used PhotolonTM. Using fluorescent microscopy, it was shown that Compound 21 quickly penetrates the cells. Analysis of caspase-3 activity indicated an apoptosis induction 40 min after exposure to red light (λ = 660 nm). The induction of DNA damages and apoptosis was shown using Comet assay. The results of expression analysis of the stress-response genes indicate an activation of the genes which control the cell cycle and detoxification of the free radicals after an exposure of HeLa cells to Compound 21 and to red light. High photodynamic activity of this compound and the ability to oxidize biomolecules was demonstrated on nuclear-free mice erythrocytes. In addition, it was shown that Compound 21 is effectively activated with low energy 700 nm light, which can penetrate deep into the tissue. Thus, Compound 21 is a prospective substance for development of the new drugs for photodynamic therapy of cancer.
Different organisms, cell types, and even similar cell lines can dramatically differ in resistance to genotoxic stress. This testifies to the wide opportunities for genetic and epigenetic regulation of stress resistance. These opportunities could be used to increase the effectiveness of cancer therapy, develop new varieties of plants and animals, and search for new pharmacological targets to enhance human radioresistance, which can be used for manned deep space expeditions. Based on the comparison of transcriptomic studies in cancer cells, in this review, we propose that there is a high diversity of genetic mechanisms of development of genotoxic stress resistance. This review focused on possibilities and limitations of the regulation of the resistance of normal cells and whole organisms to genotoxic and oxidative stress by the overexpressing of stress-response genes. Moreover, the existing experimental data on the effect of such overexpression on the resistance of cells and organisms to various genotoxic agents has been analyzed and systematized. We suggest that the recent advances in the development of multiplex and highly customizable gene overexpression technology that utilizes the mutant Cas9 protein and the abundance of available data on gene functions and their signal networks open new opportunities for research in this field.
Understanding the mechanisms producing low dose ionizing radiation specific biological effects represents one of the major challenges of radiation biology. Although experimental evidence does suggest that various molecular stress response pathways may be involved in the production of low dose effects, much of the detail of those mechanisms remains elusive. We hypothesized that the regulation of various stress response pathways upon irradiation may differ from one another in complex dose-response manners, causing the specific and subtle low dose radiation effects. In the present study, the transcription level of 22 genes involved in stress responses were analyzed using RT-qPCR in normal human fibroblasts exposed to a range of gamma-doses from 1 to 200 cGy. Using the alkali comet assay, we also measured the level of DNA damages in dose-response and time-course experiments. We found non-linear dose responses for the repair of DNA damage after exposure to gamma-radiation. Alterations in gene expression were also not linear with dose for several of the genes examined and did not follow a single pattern. Rather, several patterns could be seen. Our results suggest a complex interplay of various stress response pathways triggered by low radiation doses, with various low dose thresholds for different genes.
Морфологический и молекулярно-генетический подходы к изучению рода Dactylorhiza в Республике Коми Работа выполнена при поддержке Комплексной программы фундаментальных исследований Уральского отделения Российской академии наук на 2018 год, проект № АААА-А17-117121270031-2 (18-4-4-23) «Генетическое разнообразие редких видов Европейского северо-востока России: инвентаризация и прогноз устойчивости к глобальным изменениям климата» и частично гранта РФФИ № 16-44-110167 р_а Представлены результаты изучения видов рода Dactylorhiza Neck. ex Nevski на территории Республики Коми. На основании анализа маркерных последовательностей ДНК (ITS1-5.8S-ITS2) и 19 морфометрических признаков 480 растений изучено 16 ценопопуляций видов рода в регионе. Совместное использование морфологического и молекулярно-генетического подходов позволило подтвердить произрастание на территории Республики Коми 4 видов: Dactylorhiza fuchsii (Druce) Soό, D. maculata (L.) Soό, D. incarnata (L.) Soό s. l. (включая D. cruenta (O. F. Muell) Soó) и D. traunsteineri (Saut.) Soó. D. cruentа, повидимому, стоит рассматривать как подвид (или разновидность) D. incarnata. Приведены морфологические признаки, позволяющие разделить виды внутри рода, ими стали ширина губы и шпорца, длина прицветника, ширина нижних листьев. Получены новые молекулярно-филогенетические данные для этих видов. Вопрос о наличии D. baltica (Klinge) Orlova на территории региона остается открытым и требует дополнительных исследований.
The terrestrial environment of the East European tundra consists of a mosaic of habitat types. In addition to the natural habitat diversity, various human-influenced types may occur. In the town of Vorkuta, Komi Republic, Russia the manure-enriched soils near hydrogen sulfide springs were observed. This site represents an unusually nutrient-rich location with considerable development of organic soils, in contrast to the naturally forming soils in East European tundra which are typically thin and nutrient poor. In these organic soils, two species of Lumbricidae and two species of Collembola previously not recorded from the natural ecosystems in the study area of research territory were found. One earthworm species, Dendrodrilus rubidus tenuis, is likely to have been introduced. The presence of the three other species (Eiseniella tetraedra, Folsomia fimetaria, and Proisotoma minuta) is quite natural in East European tundra and such anthropogenic soils with high organic content may be a good habitat for them.
Molecular responses to genotoxic stress, such as ionizing radiation, are intricately complex and involve hundreds of genes. Whether targeted overexpression of an endogenous gene can enhance resistance to ionizing radiation remains to be explored. In the present study we take an advantage of the CRISPR/dCas9 technology to moderately overexpress the RPA1 gene that encodes a key functional subunit of the replication protein A (RPA). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein complex involved in DNA replication, recombination, and repair. Dysfunction of RPA1 is detrimental for cells and organisms and can lead to diminished resistance to many stress factors. We demonstrate that HEK293T cells overexpressing RPA1 exhibit enhanced resistance to cell killing by gamma-radiation. Using the alkali comet assay, we show a remarkable acceleration of DNA breaks rejoining after gammairradiation in RPA1 overexpressing cells. However, the spontaneous rate of DNA damage was also higher in the presence of RPA1 overexpression, suggesting alterations in the processing of replication errors due to elevated activity of the RPA protein. Additionally, the analysis of the distributions of cells with different levels of DNA damage showed a link between the RPA1 overexpression and the kinetics of DNA repair within differentially damaged cell subpopulations. Our results provide knew knowledge on DNA damage stress responses and indicate that the concept of enhancing radioresistance by targeted alteration of the expression of a single gene is feasible, however undesired consequences should be considered and evaluated.
The aim of our work was to obtain chloroplast (trnH-psbA) and nuclear (ITS1-ITS2) DNA nucleotide sequences and identify the phylogenetic position of Phlojodicarpus villosus (Apiaceae). This species of vascular plants is represented in the Urals by isolated relic populations and is included in the regional Red Data Books. There is no data on P. villosus nucleotide sequences in the international open genetic databases. We studied two herbarium specimens of P. villosus, one collected from the Ural part of its range in the Komi Republic (Northern Urals) and the second collected from the main part of its range in the Magadan Region (Kolyma Highlands). Combining nuclear and chloroplast markers made it possible to reliably determine phylogenetic position of P. villosus within the tribe Selineae (subfamily Apioideae, family Apiaceae). We found ITS1-ITS2 and trnH-psbA nucleotide sequences to be sufficiently informative to identify specimens of this genus. High polymorphism of P. villosus sequences obtained from different parts of its range (Northern Urals and Kolyma Highlands) and the presence of evolutionary events (deletions) require more detail study of P. villosus and other Phlojodicarpus taxa by DNA barcoding methods.
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