Biochemistry (Moscow)最新文献

Rabies is a zoonotic disease with high lethality. Most human deaths are associated with the bites received from dogs and cats. Vaccination is the most effective method of preventing rabies disease in both animals and humans. In this study, the ability of an adjuvant based on recombinant Salmonella typhimurium flagellin to increase protective activity of the inactivated rabies vaccine in mice was evaluated. A series of inactivated dry culture vaccine for dogs and cats “Rabikan” (strain Shchelkovo-51) with addition of an adjuvant at various dilutions were used. The control preparation was a similar series of inactivated dry culture vaccine without an adjuvant. Protective activity of the vaccine preparations was evaluated by the NIH potency test, which is the most widely used and internationally recommended method for testing effectiveness of the inactivated rabies vaccines. The value of specific activity of the tested rabies vaccine when co-administered with the adjuvant was significantly higher (48.69 IU/ml) than that of the vaccine without the adjuvant (3.75 IU/ml). Thus, recombinant flagellin could be considered as an effective adjuvant in the composition of future vaccine preparations against rabies virus.

The contents of homocysteine (HCy), cyanocobalamin (vitamin B12), folic acid (vitamin B9), and pyridoxine (vitamin B6) were analyzed and the genotypes of the main gene polymorphisms associated with folate metabolism (C677T and A1298C of the MTHFR gene, A2756G of the MTR gene and A66G of the MTRR gene) were determined in children at the onset of multiple sclerosis (MS) (with disease duration of no more than six months), healthy children under 18 years (control group), healthy adults without neurological pathology, adult patients with MS at the onset of disease, and adult patients with long-term MS. A significant increase in the HCy levels was found in children at the MS onset compared to healthy children of the corresponding age. It was established that the content of HCy in children has a high predictive value. At the same time, an increase in the HCy levels was not accompanied by the deficiency of vitamins B6, B9, and B12 in the blood. The lack of correlation between the laboratory signs of vitamin deficiency and HCy levels may be due to the polymorphic variants of folate cycle genes. An increased HCy level should be considered as a marker of functional disorders of folate metabolism accompanying the development of pathological process in pediatric MS. Our finding can be used to develop new approaches to the prevention of demyelination in children and treatment of pediatric MS.

Lapatinib is a targeted therapeutic inhibiting HER2 and EGFR proteins. It is used for the therapy of HER2-positive breast cancer, although not all the patients respond to it. Using human blood serum samples from 14 female donors (separately taken or combined), we found that human blood serum dramatically abolishes the lapatinib-mediated inhibition of growth of the human breast squamous carcinoma SK-BR-3 cell line. This antagonism between lapatinib and human serum was associated with cancelation of the drug induced G1/S cell cycle transition arrest. RNA sequencing revealed 308 differentially expressed genes in the presence of lapatinib. Remarkably, when combined with lapatinib, human blood serum showed the capacity of restoring both the rate of cell growth, and the expression of 96.1% of the genes expression of which were altered by the lapatinib treatment alone. Co-administration of EGF with lapatinib also restores the cell growth and cancels alteration of expression of 95.8% of the genes specific to lapatinib treatment of SK-BR-3 cells. Differential gene expression analysis also showed that in the presence of human serum or EGF, lapatinib was unable to inhibit the Toll-Like Receptor signaling pathway and alter expression of genes linked to the Gene Ontology term of Focal adhesion.

Glutathione S-transferases (GSTs) belong to the superfamily of multifunctional detoxification isoenzymes with an important role in cellular signaling. They can prevent reactive electrophilic compounds from harming the body by covalently binding identical type of moleculs to each other. GSTs can be used alone or in combination for cancer detection or diagnosis, in addition to therapeutic interventions. In recent years, indoles have become important due to their structural properties and biological activities such as antitubercular, antiulcer, anti-oxidant, and antidiabetic, as well as for the development of new anticancer agents. The current research investigated effects of some indoles with 3-carboxaldehyde structure on the GST enzyme activity. Impacts of various concentrations of indoles on the in vitro GST activity were examined. While IC₅₀ values for the compounds ranged from 0.042 to 1.570 mM, K_i values changed between 0.018 ± 0.01 and 1.110 ± 0.15 mM. 6-Methylindole-3-carboxaldehyde (1b) exhibited the highest inhibitory effect among the indoles examined. Indole derivatives used in the study can be evaluated in further pharmacological studies due to their effects on GST activity.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising agent for treatment of AML due to its specific apoptosis-inducing effect on tumor cells but not normal cells. However, emergence of resistance to TRAIL in the AML cells limits its potential as an antileukemic agent. Previously, we revealed increase in the resistance of the human AML THP-1 cells to the TRAIL-induced death during their LPS-dependent proinflammatory activation and in the in vitro model of LPS-independent proinflammatory activation – in a long-term high-density cell culture. In this study, we investigated mechanisms of this phenomenon using Western blot analysis, caspase 3 enzymatic activity analysis, quantitative reverse transcription-PCR, and flow cytometry. The results showed that the increased resistance to the TRAIL-induced cell death of AML THP-1 cells during their pro-inflammatory activation is associated with the decrease in the surface expression of the proapoptotic receptors TRAIL-R1/DR4 and TRAIL-R2/DR5, as well as with the increased content of members of the IAPs family – Livin and cIAP2. The results of this article open up new insights into the role of inflammation in formation of the resistance of AML cells to the action of mediators of antitumor immunity, in particular TRAIL.

Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST-segment elevation in the right precordial leads on electrocardiograms (ECG), and high risk of life-threatening ventricular arrhythmia and sudden cardiac death (SCD). Mutations in the responsible genes have not been fully characterized in the BrS patients, except for the SCN5A gene. We identified a new genetic variant, c.1189C>T (p.R397C), in the KCNH2 gene in the asymptomatic male proband diagnosed with BrS and mild QTc shortening. We hypothesize that this variant could alter I_Kr-current and may be causative for the rare non-SCN5A-related form of BrS. To assess its pathogenicity, we performed patch-clamp analysis on I_Kr reconstituted with this KCNH2 mutation in the Chinese hamster ovary cells and compared the phenotype with the wild type. It appeared that the R397C mutation does not affect the I_Kr density, but facilitates activation, hampers inactivation of the hERG channels, and increases magnitude of the window current suggesting that the p.R397C is a gain-of-function mutation. In silico modeling demonstrated that this missense mutation potentially leads to the shortening of action potential in the heart.

Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma.

Ionic liquids (ILs) are organic salts with a low melting point. This is due to the fact that their alkyl side chains, which are covalently connected to the ion, hinder the crystallization of ILs. The low melting point of ILs has led to their widespread use as relatively harmless solvents. However, ILs do have toxic properties, the mechanism of which is largely unknown, so identifying the cellular targets of ILs is of practical importance. In our work, we showed that imidazolium ILs are not able to penetrate model membranes without damaging them. We also found that inactivation of multidrug resistance (MDR) pumps in yeast cells does not increase their sensitivity to imidazolium ILs. The latter indicates that the target of toxicity of imidazolium ILs is not in the cytoplasm. Thus, it can be assumed that the disruption of the barrier properties of the plasma membrane is the main reason for the toxicity of low concentrations of imidazolium ILs. We also showed that supplementation with imidazolium ILs restores the growth of cells with kinetically blocked glycolysis. Apparently, a slight disruption of the plasma membrane caused by ILs can, in some cases, be beneficial for the cell.

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aβ42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer’s disease. The results showed that the Aβ42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.

The most important manifestation of aging is an increased risk of death with advancing age, a mortality pattern characterized by empirical regularities known as mortality laws. We highlight three significant ones: the Gompertz law, compensation effect of mortality (CEM), and late-life mortality deceleration and describe new developments in this area. It is predicted that CEM should result in declining relative variability of mortality at older ages. The quiescent phase hypothesis of negligible actuarial aging at younger adult ages is tested and refuted by analyzing mortality of the most recent birth cohorts. To comprehend the aging mechanisms, it is crucial to explain the observed empirical mortality patterns. As an illustrative example of data-directed modeling and the insights it provides, we briefly describe two different reliability models applied to human mortality patterns. The explanation of aging using a reliability theory approach aligns with evolutionary theories of aging, including idea of chronic phenoptosis. This alignment stems from their focus on elucidating the process of organismal deterioration itself, rather than addressing the reasons why organisms are not designed for perpetual existence. This article is a part of a special issue of the journal that commemorates the legacy of the eminent Russian scientist Vladimir Petrovich Skulachev (1935-2023) and his bold ideas about evolution of biological aging and phenoptosis.