Biochemistry (Moscow)最新文献

Primary cell cultures are one of the main research objects and promising tools in regenerative biomedicine. However, their application is significantly limited by the short lifespan and rapid aging. Existing approaches to prolong the “youth” of cultured cells inevitably alter their properties, which raises questions about their applicability in regenerative biomedicine. Our review examines the main mechanisms of aging of cultured cells, existing methods used to overcome it, and safety issues associated with the produced cultures. We analyzed the data on cell immortalization and its connection with tumor transformation. Among the methods for prolonging the proliferative activity of cells are spontaneous immortalization and immortalization induced by overexpression of the catalytic subunit of telomerase (TERT), viral oncogenes (T antigens of the polyomavirus SV40, proteins E6/E7 of human papillomavirus type 16, and adenoviral proteins E1A and E1B of adenoviruses), and cellular transcription factors, such as proto-oncogenes (c-MYC, BMI1). The accumulated data suggest that increasing the expression of the gene encoding TERT is one of the relatively safe approaches to prolonging the proliferative activity of a cell line, which does not lead to the tumor transformation of cell line. Based on the analyzed data, an attempt was made to identify the “boundary” between the permissible prolongation of cell culture life and its tumor transformation.

Starvation can initiate a programmed death in unicellular organisms. It is believed that cells committing suicide via this mechanism support surviving cells with their nutrients. It was recently discovered that heating Saccharomyces cerevisiae yeast cells to 51°C kills the cells though a special pathway that starts with the decomposition of the lysosomal membrane followed by the loss of the plasma membrane integrity a few hours later. Since lysosomes contain hydrolases of nitrogen-containing biopolymers, this programmed death pathway could help the survivors during nitrogen starvation. The loss of the plasma membrane integrity significantly alters the water/salt composition of the cytosol, which in turn can lead to the aggregation of nitrogen-containing macromolecules (proteins and nucleic acids). The pores of the yeast cell wall are too small to let such aggregates through. We showed that cells killed by heating at 52°C stimulated the growth of living cells under conditions of amino acid deficiency, while mutant cells deficient by the AP-3 complex, which were unable to destroy the lysosomal membrane, lacked this ability. Yeast cells killed by boiling also did not support the growth of surviving cells under amino acid deficiency conditions. Unexpectedly, the medium from the cells killed by boiling contained, on average, higher amounts of each amino acid than the medium from the cells killed by heating to 52°C. At the same time, according spectrophotometry and mass spectrometry analysis, the medium obtained by cell heating to 52°C contained higher amount of substances absorbing in a short-wavelength range. We speculate that the compounds supporting the growth of surviving cells under the nitrogen deficiency conditions are generated by hydrolysis of nucleic acids.

Kidney dysfunction could impose a heavy burden on patients and society by progressing to chronic kidney disease (CKD) and end-stage renal disease, which emphasizes the importance of a search for novel agents capable of slowing down kidney disease progression or ameliorating disease outcome in patients. One of the candidates for nephroprotective agents is silymarin, a flavonoid with the anti-oxidant and anti-inflammatory properties from the milk thistle plant. We performed a systematic review of articles from Scopus, PubMed, and Web of Science that compared the nephroprotective effects of silymarin in treated and control groups (studies conducted in animals or cells were excluded). We also performed a meta-analysis of changes in the serum creatinine level and ran a subgroup analysis on the silymarin dosage, treatment duration, patients’ age, and type of kidney disease. Quality assessment of the articles was performed with the ROB2 tool. We identified 10 relevant clinical trials; meta-analysis was performed on 7 studies and showed a significant effect of silymarin on the reduction of serum creatinine levels (Hedges’ g, −1.23; 95% CI, −2.02 to −0.43; p = 0.0024; I² = 93.40%). Analysis of the subgroups revealed a significant creatinine reduction in patients with the drug-induced acute kidney injury (AKI) (p = 0.003), but not with CKD (p = 0.3065). The daily silymarin dose of 280 mg was ineffective (p = 0.2375) in reducing the creatinine levels. Despite the limitations, such as a small number of analyzed articles and their heterogeneity, we found that silymarin administration in the drug-induced AKI could provide a nephroprotective effect by lowering the serum levels of creatinine.

DNA damage results in distortion of the B-form structure of the DNA double helix. Recognition of such distortion by DNA repair proteins is an important stage in the process initiation. Nucleosome structure imposes restrictions on mobility and plasticity of DNA geometry. Under interaction of repair proteins with nucleosomal DNA, the main issue is the implementation of the DNA structure that characterizes the damage itself in a specific context. In addition, the DNA duplex in a nucleosome has a regular profile of contacts with histones corresponding to the pitch of DNA helix. Changes in the DNA structure could be assessed through the changes in this profile. This profile correlates with availability of the corresponding nucleotides for interaction with the DNA-binding proteins. In our work, it has been shown using footprinting assay that the presence of an AP site within the second or third turn from the 5′-end of the nucleosomal DNA does not significantly affect the profile of DNA contacts with histones.

The goal of this study was examination of the association between the expression levels of the genes involved in high-density lipoprotein metabolism and atherogenesis and underlying metabolic pathways and the number of stenotic coronary arteries. Expression of 65 preselected genes in the peripheral blood mononuclear cells of the control patients (n = 63) and patients with coronary artery disease (CAD) with one or two (low stenosis group, n = 35) or three or four (high stenosis group, n = 41) stenotic vessels, confirmed by coronary angiography, was measured with real-time PCR. Functional enrichment analysis was applied for annotation of differentially expressed genes. Protein products of the differentially expressed genes (DEGs) in the CAD patients compared to the controls were associated with metabolic pathways related to assembly, remodeling, and clearance of plasma lipoproteins, as well as with signaling and regulation of expression of the genes involved in cholesterol transport and efflux. However, comparison of the gene expression profiles and associated metabolic pathways between the groups with high versus low stenosis revealed specific differences between these groups. Expression of the CETP, PLTP, CD36, IL18, ITGB3, S100A8, S100A12, and VEGFA genes increased with the increase of the number of stenotic vessels, which suggests involvement of these genes in stenosis expansion via lipoprotein metabolism, inflammation, angiogenesis, and innate immunity. The set of genes ITGB3, VEGFA, and CETP was selected as a new gene expression signature of expansion of the coronary artery stenosis, which was validated with the GSE12288 dataset from the Gene Expression Omnibus database, demonstrating an average odds ratio (OR) of 7.49 (95% CI, 2.21 to 25.43). Averaged expression levels of the ITGB3, VEGFA, and CETP genes could be used for diagnosis, prognosis evaluation, and treatment of coronary stenosis with strong predictive power.

The deficiency of glucocerebrosidase (GCase) encoded by the GBA1 gene, leads to the autosomal recessive Gaucher disease and highly increased risk of developing Parkinson’s disease (PD). In order to study the effect of GCase dysfunction on neurodegeneration, we evaluated the GCase activity, lysosphingolipid content, extent of dopaminergic neuron degeneration in the substantia nigra (SN), and levels of dopamine (DA) and total and oligomeric α-synuclein (α-Syn) in the brain of mice with the presymptomatic stage of parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in combination with a single injection of the GCase selective inhibitor conduritol-β-epoxide (CBE) (100 mg/kg body weight). A single injection of CBE led to a ~50% decrease in the GCase activity, significant increase in the lysosphingolipid content, and striatal accumulation of oligomeric α-Syn in the mouse brain. Assessment of the DA neuron degeneration in the SN 14 days after injection by immunohistochemical staining for tyrosine hydroxylase (TH) demonstrated a twice more pronounced reduction in the number of TH+ neurons in MPTP+CBE mice compared to MPTP only-treated animals (14% vs. 29%, respectively; p < 0.0001). The double neurotoxic (MPTP+CBE) model was also characterized by a decrease in the DA content and more pronounced accumulation of total α-Syn in the striatum. Overall, we demonstrated that inhibition of the GCase activity leads to the α-Syn accumulation and further exacerbation of the MPTP-induced pathology. The described double toxic MPTP+CBE mouse model can be used for the screening of neuroprotective drugs in approaches aimed at increasing the GCase activity.

Aging biomarkers could potentially facilitate assessment of the rate of aging, age-related diseases, and allow monitoring of the effectiveness of preventive interventions such as diet, physical activity, and geroprotectors. This article examines key criteria for the aging biomarkers, including their association with age, prognostic value regarding mortality, ability to detect early stages of age-related pathologies, and minimal invasiveness. Comprehensive classification of the markers (clinical, functional, molecular, omics, digital) and evolution of “aging clocks” – from epigenetic models to causal systems based on Mendelian randomization – is presented. Special attention is given to the explainable artificial intelligence technologies that allow for interpretation of the algorithms, as well as practical use of the biomarkers in clinical studies in the manner understandable to humans. Prospects for the development of the comprehensive panels of biomarkers and personalized approaches to aging assessment are discussed.

Ribosomes are macromolecular machines of conveyor type, which move along the mRNA from triplet to triplet and polymerize cognate amino acids. They are regarded as uniform entities with constant molecular composition bearing no regulatory capacity. However, their ability to interact with multiple proteins involved in translation suggests existence of specialized ribosomes dedicated to biosynthesis of particular proteins. This can be easily imagined in yeast mitochondria, whose genomes encode eight polypeptides, and where protein-specific translation is already represented by translational activators – a group of proteins each regulating a single mRNA translation. Despite this, the subject remains poorly investigated. We report an exploratory approach to search for distinct populations of ribosomes in the yeast mitochondria. Affinity tags were added to mitochondrial ribosomal proteins, ribosomes were isolated by immunoprecipitation and their protein and RNA content was characterized. It was shown that the mitochondrial ribosomes isolated from yeast strains bearing affinity tags on different ribosomal proteins recover different sets of components. The differences were rather quantitative than qualitative, because almost full sets of mitochondrial ribosomal proteins were identified in each sample, but the ratios demonstrated variance ranging from 10 to less than 0.05 molecules per ribosome. In addition, we explored the power of translational activators as a bait to recover ribosomes translating specific mRNAs in yeast mitochondria.

Endocrine disorders represent a serious public health problem and frequently can be caused by genetic factors or their combination with environmental and lifestyle factors. Assessment of relevant genetic factors is important to estimate the risk of endocrine pathologies in an individual before their manifestation. Identification of genetic variations in proteins of the major histocompatibility complex is important with regard to the autoimmune nature of many endocrine pathologies, including type 1 diabetes. In this study, we investigated the relationship between human leukocyte antigen (HLA) genes and 13 endocrine disorders by using experimental whole-exome sequencing profiles obtained for 895 patients from the National Medical Research Center for Endocrinology, Moscow. In addition, the linkage disequilibrium of the identified alleles in the context of the respective diagnoses was assessed. We identified totally 45 statistically significant associations between HLA alleles and specific diagnoses of endocrine pathologies. Among them, 33 were described for the first time and 12 have been previously reported for type 1 diabetes. Overall, 17 alleles were associated with type 1 diabetes and four alleles – with other forms of diabetes. Furthermore, three alleles were associated with obesity, five – with adrenogenital diseases, three – with hypoglycemia, and three – with precocious puberty. Single alleles were found to be associated with congenital hypothyroidism without goiter, hyperfunction of pituitary gland, adrenomedullary hyperfunction, and short stature due to endocrine disorder. The study shows that early HLA typing can help in detecting genetic risk factors of endocrine disorders. In addition, identification of disease associations with specific HLA alleles can broaden our understanding of the mechanisms involved in the pathogenesis of relevant endocrine disorders.

Multiple drug resistance is one of the major threats to global health. One of the approaches to solving this problem is antimicrobial photodynamic therapy, however, currently used photosensitizers are not sufficiently effective against pathogens. Polycationic molecular constructs enhance the binding and penetration of photosensitizers into poorly permeable gram-negative bacteria. Such conjugates can be obtained by introducing polyethyleneimines into a photosensitizer molecule. In this study, we synthesized a branched tetraamine and introduced it into the pyrrole ring A of the natural chlorin molecule and assessed in vitro the photoinduced toxicity of the new photosensitizer against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli bacteria. Compared to its structural precursor, the obtained chlorin with a branched polyamine residue demonstrated an increased bactericidal effect when irradiated with light.