Doklady Biochemistry and Biophysics最新文献

In the previously created modular nanotransporter (MNT) capable of delivering a monobody to Keap1 into the cytosol, the endosomolytic module, translocation domain of diphtheria toxin (DTox), was replaced by the endosomolytic peptide GALA3. It was found that this substitution more than doubles the lifetime of MNT in the blood. Using confocal microscopy, it was shown that MNT with GALA3 was internalized into AML12 cells mainly due to binding to the epidermal growth factor receptor, and is also able to exit from endosomes into the cytosol. Using cellular thermal shift assay, it was shown that MNT with GALA3 and MNT with DTox are equally effective in disrupting the formation of the Nrf2 complex with Keap1, which led to similar protection of AML12 cells from the action of hydrogen peroxide. The obtained results allow not only optimizing the systemic use of MNT, but can also serve as a basis for creating agents aimed at treating diseases associated with oxidative stress.

In this work, two new compounds, N⁶-(4,5-dimethoxyphenyl)adenine and N⁶-(3,5-di-trifluoromethylphenyl)adenine, with a broad range of antiviral activity against RNA viruses were identified. We showed that these compounds exhibit pronounced antiviral activity against human poliovirus types 1, 2, and 3, belonging to enterovirus C species. Both compounds also demonstrated pronounced antiviral activity against Coxsackie viruses B3, B5, and B6, belonging to enterovirus B species. In addition, the compounds demonstrated antiviral activity against Newcastle disease virus, which belongs to the paramyxovirus genus. The compounds discovered in this work can subsequently serve as prototypes for the development of new antiviral drugs against epidemiologically significant human RNA viruses.

Graves’ disease is caused by overactivation of the thyroid-stimulating hormone receptor (TSHR). One approach for its treatment may be the use of negative allosteric modulators (NAM) of TSHR, which normalize TSHR activity and do not cause thyroid hormone (TH) deficiency. The aim of the work was to study the effect of a new compound 5-amino-4-(4-bromophenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxylic acid N-tert-butylamide (TPY4) on the basal and TSH-stimulated TH production in cultured FRTL-5 thyrocytes and on basal and thyrotropin-releasing hormone (TRH)-stimulated TH levels in the blood of rats. TPY4 stimulated TH production by thyrocytes and increased TH levels when administered intraperitoneally and orally in rats. It also decreased the TSH-stimulated TH production in thyrocytes and the TRH-stimulated TH levels in rats. Thus, TPY4 is the first known allosteric regulator of TSHR, combining the properties of NAM and a partial agonist, and can be considered as a prototype of drugs for the treatment of Graves’ disease.

Introduction. Breast cancer resistance protein (BCRP) is an efflux membrane transporter that controls the pharmacokinetics of a large number of drugs. Its activity may change when taking some endo- and exogenous substances, thus making it a link in drug interactions. Aim. The aim of the study was to develop a methodology for testing drugs for belonging to BCRP substrates and inhibitors in vitro. Materials and methods. The work was performed on Caco-2 cells overexpressing BCRP, the cultivation was performed in a transwell system consisting of the apical and basolateral chambers. Cells were seeded at the bottom of the apical chamber, which is a semi-permeable membrane. Primarily, the transport of BCRP substrates—methotrexate, mitoxantrone, and quercetin—was evaluated in the concentration range of 1, 5, 10, and 50 μM in the direction from the basal chamber to the apical one (Papp b-a) and in the opposite direction (Papp a-b). The Papp b-a/Papp a-b ratio more than 2 characterizes the involvement of transporter proteins in the transcellular transport of substances. To confirm the involvement of BCRP in their transport, an experiment was carried out with the addition of a transporter inhibitor, reserpine, at a concentration of 50 μM to the transport medium. The concentration of substrates in the chambers was analyzed by HPLC–MS/MS. Results. After the addition of methotrexate (1 μM), mitoxantrone (1 μM), and quercetin (1–10 μM) to the apical or basolateral chambers of the transwell system, their content in the recipient chamber was not detected. At methotrexate concentration of 5 μM, the Papp b-a/Papp a-b ratio was 3.38 ± 0.08, which indicates the involvement of transporters in its transfer. When methotrexate was added to the donor chamber at concentrations of 10 and 50 μM, the Papp b-a/Papp a-b ratio decreased to values below 2. At mitoxantrone concentration of 5 μM, the Papp b-a/Papp a-b ratio was 2.72 ± 0.16. An increase in the concentration to 10 μM led to an increase in the Papp b-a/Papp a-b ratio to 6.18 ± 0.08. At the substance concentration of 50 μM, the index decreased but remained above 2. At the quercetin concentration of 50 µM, the Papp b-a/Papp ratio was below 2. Reserpine reduced the Papp b-a/Papp a-b ratio of methotrexate 3.31 times (p = 0.0002), which indicates the elimination of asymmetry in the transport of the substance. At a mitoxantrone concentration of 10 µM, reserpine reduced its Papp b-a/Papp a-b ratio 3.36 times (p < 0.0001). These results indicate the involvement of BCRP in the control of the transfer of both substances through the cellular monolayer. Conclusions. A method of testing drugs for affiliation to BCRP substrates and inhibitors using methotrexate (5 μM) and mitoxantrone (10 μM) as marker substrates and reserpine (50 μM) as inhibitor was developed and tested on Caco-2 cells.

It was previously shown that the original dipeptide mimetic of the 4th loop of neurotrophin-3 (NT-3) hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301), like the full-length neurotrophin, predominantly activates the tyrosine kinase receptor TrkC and has a neuroprotective effect in vitro at concentrations of 10^–5–10^–12 M, as well as antidiabetic (0.1 and 0.5 mg/kg) and antidepressant (5 and 10 mg/kg) effects after systemic administration in rodents. In this work, the analgesic properties of GTS-301 were identified, which were manifested in the dose range of 0.01–10 mg/kg after acute intraperitoneal injection to rats in the “tail flick” test. Dipeptide GTS-301 increased the threshold of pain response by 20–30%; this effect persisted for at least 24 h after administration.

The expression profile of the key genes of biosynthesis (VTC2, GPP, GalDH, and GalLDH) and recycling (MDHAR1, MDHAR4, and MDHAR5) of ascorbate in response to infection with the fungal pathogen Fusarium proliferatum in garlic cultivars resistant (Podnebesny) and sensitive (Dubkovsky) to Fusarium rot was determined. It was found that differences in resistance to Fusarium lead to discrepancies in the dynamics and expression of individual genes of the ascorbate pathway, as well as in the ascorbate content. It was shown that, in response to infection, the expression level of the MDHAR4 gene increases in the resistant cultivar and decreases in the Fusarium-sensitive accession. As infection progresses, the expression levels of the VTC2 and GalLDH genes increase significantly (higher in the cv. Dubkovsky than in the cv. Podnebesny). In both cultivars, the ascorbate content increases (1.5 times higher in the cv. Dubkovsky than in the cv. Podnebesny).

The association of the pathogenesis of neurodegenerative diseases, depression, anxiety, and cognitive disorders with neurotrophin-3 deficiency determines the prospect of creating drugs with a similar mechanism of action. Since the use of full-length NT-3 is limited by unsatisfactory pharmacokinetic properties, the creation of low-molecular mimetics of neurotrophin-3 that are active when administered systemically is relevant. The Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies has created a dimeric dipeptide mimetic of the 4th loop of NT-3, hexamethylenediamide bis-(N-γ-oxybutyryl-L-glutamyl-L-asparagine) with the laboratory code GTS-302, which activates TrkC and TrkB receptors. Purpose. The purpose of the work was to study the range of pharmacological activity of GTS-302. Materials and methods. The pharmacological effects of GTS-302 were revealed by its intraperitoneal administration. The antidepressant-like activity of GTS-302 was studied in the forced swimming test on mice after its acute and 7-day administration. The anxiolytic and memory-enhancing activities of the dipeptide were studied, respectively, in the elevated plus maze on mice and in the novel object recognition test on rats after acute administration. The effect of GTS-302 on pain sensitivity was studied in the hot plate test on mice after acute administration. Results. It was found that GTS-302 exhibits antidepressant-like activity upon acute administration at doses of 0.5, 1.0, 5.0, and 10 mg/kg. At 7-day administration, the antidepressant-like activity of GTS-302 was more pronounced in terms of the effect expression and statistical significance. Dipeptide GTS-302 at doses of 1.0, 5.0, and 10.0 mg/kg exhibited anxiolytic and memory-enhancing activity and did not affect pain sensitivity. Conclusions. The pharmacological spectrum of the low-molecular NT-3 mimetic dipeptide GTS-302, revealed during systemic administration, includes a number of neuropsychotropic effects characteristic of a full-size neurotrophin. This allows GTS-302 to be considered as a potential neuropsychotropic drug.

The aim of this study was to describe the features of myocardial lymph flow using a new combined method of visualization of the lymphatic system. The study was performed on pig hearts harvested from a local slaughterhouse. The original dye, consisting of lipid-soluble chlorophyll and lipiodol, was injected stepwise into the lymphatic vessels. After sufficient optical identification of the lymphatic vessels, continuous injection of air into the coronary arteries was performed and CT scans were done. In this way, both optical and radiologic visibility of the cardiac lymphatic system was achieved. It was shown that lymph flow of the left and most part of the right ventricle is carried out through lymphatic collectors of the anterior wall of the heart, including retrogradely with respect to the right coronary artery, which complements the previously known facts about the structure of the lymphatic system of the heart.

Background. The effects of ionizing radiation (IR) involve a highly orchestrated series of events in cells, including DNA damage and repair, cell death, and changes in the level of proliferation associated with the stage of the cell cycle. A large number of existing studies in literature have examined the activity of genes and their regulators in mammalian cells in response to high doses of ionizing radiation. Although there are many studies, the research in effect of low doses of ionizing radiation remains limited. Though much progress has been made in understanding the basic principles of effects of low doses of radiation on individual components of biological systems, less is known about how low doses affect target molecules and regulate the cellular networks (e.g., activation of the immune system, genes and their regulators in the phenomenon of hormesis, and the formation of an adaptive response). These observations determined the purpose of the work: to investigate the activity of genes and non-coding RNAs (long non-coding RNAs and microRNAs) in various organs of mice with transplanted Lewis carcinoma after low-dose radiation. Materials and methods. Twenty-four female C57Bl/6 mice were transplanted subcutaneously with Lewis carcinoma cells (10⁵ cells in 0.2 mL of Hanks’ solution). Total 4-fold X-ray irradiation with an interval of 4 days at a dose of 0.075 Gy (0.85 Gy/min) was performed on the RUST M1 from 6 days after transplantation; the tumor size was measured daily. The mice were divided into the following groups: “Biocontrol”, “Biocontrol + irradiation”, “Tumor” and “Tumor + irradiation”. On the 19th day from the beginning of the experiment, the mice were euthanized. The expression profiles of mRNA genes, long non-coding RNAs, and microRNAs controlling the response to radiation were determined in the bone marrow, thymus, spleen, and tumor of mice. Results. Fractionated low-dose irradiation of mice with transplanted Lewis carcinoma caused a growth decrease of implanted tumor cells compared to the similar group without irradiation. At the same time, there was an activation of oncosuppressors and a decrease in the activity of oncogenes in the thymus and spleen of mice with tumor and irradiation. In the “Tumor” group, without irradiation, the number of activated oncogenes prevailed over the number of inactivated ones. Conclusions. Thus, the low-dose radiation exposure led to the activation of antitumor immunity in mice, which manifested itself in slowing tumor growth in animals and the induction of oncosuppressors and inhibition of oncogene expression.