The study demonstrates the possibility of using starch to model macromolecular crowding and intracellular medium. A kinetic approach is used to reveal specific functioning of butyrylcholinesterases from different sources in the starch medium. In the study solutions, the Michaelis constant increases, indicating a decrease in the enzyme affinity for the substrate in the presence of the polymer. In the 9
In this study, we examined changes in hormonal status, oxidative status, antioxidant defense, and peripheral blood leukocyte telomere length in male Javanese macaques (Macaca fascicularis) following transfer from group housing to individual cages as a model of social isolation. The study was performed in ten male Javanese macaques aged 11–17 years using a longitudinal design: the parameters were assessed in the same animals before transfer to individual cages and after 60 days of isolation. Malondialdehyde (MDA) concentration, superoxide dismutase (SOD) and catalase activities, cortisol, testosterone, and thyroglobulin levels, as well as absolute mean telomere length, were measured. A statistically significant decrease in telomere length (p = 0.0062), MDA concentration, and catalase activity (p < 0.01), as well as an increase in SOD activity and cortisol level (p < 0.05), was observed. Testosterone showed a decreasing trend, whereas thyroglobulin showed an increasing trend. The obtained data indicate systemic changes in the studied parameters in response to isolation. Changes in telomere length and oxidative status may reflect the involvement of mechanisms related to DNA damage and antioxidant system function. The results are consistent with the concept of stages of the general adaptation syndrome.
It is well established that the early stage of mechanical unloading in both humans and animals is characterized by a decline in the activity of the key cellular energy sensor, AMP-activated protein kinase (AMPK). This decline triggers signaling pathways that drive myosin phenotype remodeling and muscle atrophy. However, the molecular mechanisms underlying the reduction in AMPK activity under these conditions remain poorly understood. We hypothesized that elevated activity of protein kinase D1 (PKD1) could contribute to the decline in AMPK activity during the initial stage of rat hindlimb unloading. The present study demonstrates that the suppression of upregulated PKD1 activity via a specific inhibitor (CRT0066101) during 24-hour unloading results in the restoration of AMPK activity in the rat soleus muscle. Thus, we have shown for the first time the contribution of PKD1 to the regulation of AMPK activity in the mammalian postural muscle during the initial stage of hindlimb unloading.
A study was conducted to examine the effects of visible red light phototherapy with a wavelength of 635 nm on the expression of the neuronal protein GAP-43 in the hippocampus of rats. It was shown that the use of PBMT promotes increased GAP-43 synthesis in the hippocampus, with the maximum effect developing by the 6th day of use, which may indicate a cumulative effect of this procedure.
Nuclear export of mRNA is one of the key stages of gene expression in eukaryotes. A wide range of mRNAs is exported by the TREX-2 complex, which includes the PCID2 RNA-binding protein. Previously, we showed that PCID2 is responsible for specific recognition of transcripts, and we identified its binding site on the Drosophila ras2 gene mRNA within the 3'-non-coding region. However, the interaction was not localized for other mRNAs, which makes it impossible to identify the general patterns of PCID2 interaction with mRNA. In this work, we investigated the interaction of PCID2 with the mRNA of the kruppel gene of Drosophila melanogaster. In the EMSA experiments, we showed that PCID2 binds to two fragments of kruppel mRNA, from the 5' non-coding and coding mRNA regions. Thus, the PCID2 binding site can be located not only in the 3'-non-coding region, as in the case of the ras2 mRNA, but also in other mRNA regions. PCID2 has a lower affinity for binding sites in the kruppel mRNA than in the ras2 mRNA. Our analysis of the mRNA structure at three binding sites led to a general interaction model, in which a hairpin-type conformation emerges as the most probable structural motif at these sites.
Triple-negative breast cancer (TNBC) is characterized by an aggressive clinical course and extremely limited targeted therapy options. One of the key drivers of immunosuppression in the tumor microenvironment is the polarization of tumor-associated macrophages (TAMs) towards the M2 phenotype. In the present study, we investigated the effect of insulin-like growth factor 2 (IGF2)-the primary ligand of the IGF1R receptor and a target of the inhibitory protein IGFBP6-on the transcriptional profile of macrophages polarized in vitro from the THP-1 cell line. We demonstrate that IGF2 exerts fundamentally different effects depending on the baseline functional state of the macrophages: in immature (M0) and M2-polarized cells, it induces the expression of pro-inflammatory genes, whereas in M1 macrophages it triggers pronounced immunosuppressive reprogramming. These findings suggest that IGFBP6, by inhibiting IGF2, may sustain an anti-tumor immune response within the TNBC microenvironment.
The study proposes an experimental approach to determining the rates of individual stages of a reaction catalyzed by bacterial luciferase, based on the tryptophan fluorescence of the protein and the stopped-flow technique. The relationship between the fluorescence intensity of tryptophan residues in luciferase and the presence of substrates and reaction products in the active site of the enzyme is substantiated. The non-steady-state kinetics of bioluminescence in the reaction of two bacterial luciferases with aliphatic aldehydes of different chain lengths, as well as the kinetics of enzyme fluorescence intensity during the reaction, were analyzed. The obtained results confirmed the relationship between the rate of the two kinetic stages of enzyme luminescence and the processes of flavin substrate binding and enzyme activity recovery after the catalytic act.
The effect of a pencil scanning proton beam in two regions of the Bragg curve with different linear energy transfer (LET) relative to X-ray radiation on the induction of micronuclei (MN) in cytochalasin-blocked binucleate lymphocytes (CBBLs) during in vitro irradiation of human peripheral blood at doses ranging from 0 to 2.0 Gy was studied. A nonlinear dose-response relationship was observed in the dose range of 0 to 1.0 Gy. The frequency of MN in CBBLs during proton irradiation was 2 times lower than during X-rays and did not depend on the LET value. In the dose range above 1 Gy, the dose dependences were linear, the value of the relative biological effectiveness depended on LET and was equal to 0.76 before the Bragg peak, and 1.16 at the peak.
The SAGA complex is an important player in the regulation of eukaryotic gene transcription. The interaction of SAGA with the TREX-2 mRNA nuclear export complex makes it possible to efficiently transcribe and export mRNA at loci of actively transcribed genes near nuclear pores. However, it remains unclear whether TREX-2 is involved in anchoring the SAGA complex to the nuclear pore. In this work, we investigated the effect of knockdown of the TREX-2 complex subunits, ENY2 and Xmas-2, on the localization of the SAGA catalytic subunit, histone acetyltransferase Gcn5, in Drosophila melanogaster. The effect of knockdown on the distribution of Sgf11, a component of the SAGA deubiquitinating module, and an mRNP particle component, was also studied. We demonstrated that knockdown of ENY2 and Xmas-2 does not affect the localization of Gcn5 and Sgf11 in proximity to the nuclear pore. Therefore, TREX-2 is not required for the association of SAGA with the nuclear pore. Meanwhile, knockdown of ENY2 and Xmas-2 results in nuclear accumulation of Sgf11, suggesting that TREX-2 is involved in the localization of the SAGA-independent fraction of Sgf11 that participates in mRNA export within the mRNP particle.
The design of prodrugs for nucleoside antiviral agents is the primary approach to achieving target profiles not only in terms of safety but also efficacy. We have proposed a new chemotype of inhibitors of tick-borne encephalitis virus (TBEV) reproduction among N6-substituted adenosine derivatives, characterized by the presence of a hydrophobic aromatic substituent at the N6 position of adenine and benzoic acid residues attached via ester bonds to the ribofuranose scaffold. The compounds inhibited TBEV reproduction at micromolar concentrations only in their benzoylated form, while exhibiting no detectable cytotoxicity regardless of the substitution on the ribofuranose moiety. Our study demonstrates that modifying nucleoside analogs with hydrophobic groups can yield compounds with improved antiviral activity, and the identified compounds may subsequently serve as prototypes for the development of new antiviral drugs against epidemiologically significant pathogenic RNA viruses affecting humans.
Effective gene therapy depends on efficient delivery of genetic material into cells. Polyplexes, complexes of positively charged polymers and DNA, are a common non-viral delivery system. In this study, polyplexes were formed using a plasmid encoding the yellow fluorescent protein, TurboYFP, and polyethylenimine-polyethylene glycol-(TAT peptide) (PEI-PEG-TAT) block copolymers. These were used to transfect human lung adenocarcinoma A549 cells. Results showed that increasing the PEI nitrogen to DNA phosphate ratio and/or increasing the polyplex concentration significantly improved transfection efficiency, from a few percent up to 100 percent. These findings are valuable for producing specific proteins in cells, which may have applications in research and in the treatment of various diseases.
Polypeptide modular nanotransporters (MNTs) were engineered as a targeted delivery platform for prostate cancer cells. The constructs integrate a ligand module to facilitate specific cellular binding and internalization, and a nuclear localization signal (NLS) to enable subsequent nuclear translocation. Two distinct ligand modules were utilized: (a) a nanobody targeting prostate-specific membrane antigen (anti-PSMA) and (b) a gastrin-releasing peptide (GRP) fragment targeting the GRP receptor (GRPR). It was shown that all modules within MNT-antiPSMA and MNT-GRP retained their functional properties. The MNT-antiPSMA construct demonstrated the capacity to accumulate specifically in the nuclei of prostate cancer cells with both low and high PSMA expression. Conversely, MNT-GRP exhibited selective nuclear entry exclusively in cells characterized by high GRPR expression.
Aliphatic aldehydes are involved in many important biological processes, but detection of them at low concentrations requires expensive laboratory equipment and labor-intensive analytical methods. Typically, chromatographic and chromatograph mass spectrometric methods are used for this purpose. Therefore, developing a simple method for detecting aldehydes at low concentrations is a pressing scientific and technical challenge. We have demonstrated that a bacterial bioluminescent system can be effectively used for the semiquantitative determination of biogenic aldehydes in vitro at nanomolar concentrations. Furthermore, this system holds promise for detecting biogenic aldehydes in vivo.
Effective approaches for selecting nuclei in which a gene of interest is in an active state are necessary for studying spatial organization and gene expression regulation. In this work, a novel two-component genetic system was created for the parasegment-specific labeling of nuclei in which the regulatory domain iab-5, which stimulates the homeotic gene Abd-B, is active. The system is based on the integration of a transgene expressing the yeast GAL4 activator under the control of the minimal hsp70 gene promoter into the iab-5 domain, near the early embryonic enhancer. As a result, the iab-5 enhancer induces specific expression of GAL4, which strongly amplifies the expression of the mScarlet fluorescent protein, allowing for the efficient selection of labeled nuclei in which the iab-5 domain is activated. This approach can be used for the selection of target nuclei in which any regulatory element or gene of interest is in an active or repressed state.
The patterns of radiation adaptive response (RAR) induction and transgenerational genomic instability in mice following exposure to carbon ions (12C) with a linear energy transfer (LET) of 39 keV/μm and to X‑rays with a LET of 2 keV/μm at a dose of 10 cGy were studied. Low doses of 12C, as well as X‑rays, induce RAR, the magnitude of which depends on the quality of the challenge radiation. In the first- and second- generation offspring of males irradiated 12C at a dose of 10 cGy, an increased spontaneous level of cytogenetic damage and the absence of RAR in the first generation were detected, in contrast to the offspring of males after irradiation with X‑rays.
Boron Neutron Capture Therapy (BNCT) is one of the innovative methods for treating oncological diseases. Its selectivity is based on the targeted delivery of the boron-10 isotope to tumor cells, followed by neutron irradiation, the 10B(n, α)7Li reaction occurs with a local release of 2.79 MeV of energy. Budding boron delivery agents are nanoscale systems. This study evaluated in vitro cytotoxicity, accumulation, and retention of elemental boron nanoparticles, synthesized by laser ablation and laser fragmentation, in U87 and BT474 tumor cells and BJ-5ta fibroblasts. It was shown that both types of nanoparticles exhibit low cytotoxicity at therapeutically relevant concentrations. Boron accumulation was maximal after 24 h of incubation and was significantly higher in tumor cells, especially in the BT474 cell line, compared to fibroblasts. The obtained data indicate the promise of these nanoparticles as boron delivery agents for BNCT.
The lipid envelope of the influenza A virus contains two major types of protein spikes formed by the transmembrane hemagglutinin trimers (HA, MW 80 kDa) and neuraminidase tetramers (NA, MW 55 kDa), in quantities of 500 and 120, respectively. The third transmembrane protein, M2 (MW 14 kDa), forms tetramers of ion channels in quantities of about 10-20 per virion. Modeling of the molecular structure using the AlphaFold software tool showed a novel model for a heterocomplex of HA0-M2 proteins, in which the M2 tetramer located inside the HA0 trimer like a "matryoshka doll." Similar models of the HA0-M2 heterocomplex were obtained for the A/Aichi/2/68 (H3N2) and A/WSN/33 (H1N1) viruses. The resulting HA0-M2 heterocomplex possessed a high structural complementarity of the macromolecular interfaces (ipTM = 0.65), had no structural clashes of atoms in the molecular interfaces (clash score = 0.0), and exhibited stable and reliable intermolecular topology with a high ranking score of 0.79. The constructed model allows to explain the phenomenon of blocking the function of M2 ion channels by the rigid conformation of the uncleaved HA0 protein and, in contrast, the activation of M2 channels after (i) specific point proteolysis of HA0 into HA1 (55 kDa) and HA2 (25 kDa) subunits and (ii) exposure to acidic pH of 4.0-5.5, leading to the disclosure of the 3D structure of the HA1/2 molecule and the opening of the M2 channel.
We previously demonstrated that fragments of the central loop of the non-conventional toxin WTX reduce blood pressure in rats and inhibit certain nicotinic acetylcholine receptor subtypes [10]. In the present study, we examined the effects of hexamethonium and methylicaconitine, which are nicotinic acetylcholine receptor inhibitors, as well as of atropine, a muscarinic acetylcholine receptor inhibitor, on the hypotensive effect of the WTX central loop fragment. For this purpose, these compounds were administered intravenously before the peptide. We found that hexamethonium and methylicaconitine enhanced the fragment's hypotensive effect, while atropine weakened this effect. Only methylicaconitine enhanced the tachycardic effect of the peptide. These data indicate the involvement of both nicotinic and muscarinic acetylcholine receptors in the hemodynamic effects of the WTX central loop fragment.
Trans-splicing is a rare variant of the splicing process, wherein exons are joined between different pre-mRNAs. This mechanism enables the formation of a broad spectrum of mRNAs encoded by a single locus. The most prominent example in Drosophila melanogaster is the mod(mdg4) locus, where trans-splicing results in the production of more than 30 mRNAs encoding protein isoforms with distinct C-terminal domains. Previous studies have demonstrated that sequences in the 5'-region of the fourth intron of the mod(mdg4) locus play a pivotal role in initiating trans-splicing. In the present study, we show that the integration of MS2 repeats, which form a stable secondary RNA structure, into the sequence of the fourth intron completely suppressed trans-splicing. Conversely, the insertion of MS2 repeats immediately upstream of the intron only marginally reduced the efficiency of this process. Modifications of the sequences of the fourth intron that induce the formation of an additional stem loop in the pre-mRNA also negatively impacted trans-splicing. These findings underscore the significant role of RNA secondary structures in regulating trans-splicing.
