[Pathogenetic basis of optic nerve atrophy in methanol poisoning]. / Patogeneticheskie osnovy razvitiya atrofii zritel'nogo nerva pri toksicheskom porazhenii metanolom.

Optic nerve atrophy is a pathomorphological consequence of diseases of the peripheral neuron of the visual pathway, manifested as atrophy of nerve fibers of varying severity. The toxic effect of methanol is mainly associated with formic acid and formaldehyde, which suppress the cytochrome system, inhibit oxidative phosphorylation, and thereby cause a deficiency of adenosine triphosphoric acid, to which brain and retinal tissues are especially susceptible. When formiate accumulates, tissue respiration is disrupted, leading to pronounced tissue hypoxia. As a result of such methanol metabolism, metabolic acidosis occurs. Tissue hypoxia develops in the first few hours as a result of the action of formic acid on the respiratory enzyme chain at the cytochrome oxidase level. Hypoxia and, as a consequence, a decrease in energy supply lead to a disruption of biological oxidation and the development of apoptosis in the optic nerve fibers. Understanding the process of optic nerve atrophy development at the pathogenetic level in methyl alcohol intoxication will help make a correct early diagnosis and prescribe timely treatment.

[Stroke in children: experience of the Center for the Treatment of Cerebrovascular Diseases in Children and Adolescents in Moscow]. / Insul't u detei: opyt raboty Tsentra po lecheniyu tserebrovaskulyarnykh zabolevanii u detei i podrostkov Moskvy.

OBJECTIVE: To analyze the work of the Center for the Treatment of Cerebrovascular Pathology in Children and Adolescents, operating on the basis of the Morozov Children's City Clinical Hospital of the Moscow Health Department for the period 2018-2021 and to assess the peculiarities of organizing the provision of specialized care to children and adolescents with acute cerebrovascular accident (ACA). MATERIAL AND METHODS: Annual reports of the Center for the period 2018-2021; included children and adolescents aged 1 month to 17 years 11 months 29 days, with new onset ischemic stroke (IS) and hemorrhagic stroke (HS), cerebral venous thrombosis (sinus thrombosis), confirmed clinically and radiologically. RESULTS: Statistical data on stroke and organization of care for children with this pathology in Moscow are presented. The incidence of IS in Moscow for the period 2018-2021 ranged from 1.6 to 2.5 per 100.000 children, HI - from 2.35 to 3.3 per 100.000, sinus thrombosis from 0.5 to 1.38 per 100.000. When assessing the main etiological factors of stroke in The Center for International Pediatric Stroke Research categories, we noted a prevalence of chronic head and neck diseases (20-37%) and chronic systemic conditions (conditions or diseases with known changes in coagulation or vascular structure, including connective tissue dysplasia, genetic, hematological, inflammatory or diseases of the immune system) (15-20%). In addition, data on reperfusion therapy carried out at the Center are presented. From 2018 (first thrombolysis was performed) to 2021, 7.3-14.7% of all patients with IS underwent thrombolysis. CONCLUSION: The experience of functioning of the Center for the Treatment of Cerebrovascular Pathology in Children and Adolescents has shown that the creation of such centers in the regions of the Russian Federation is relevant, but requires taking into account the characteristics of the pediatric population when organizing their work.

[Comparative Analysis of Family A DNA-Polymerases as a Searching Tool for Enzymes with New Properties].

DNA polymerases catalyze DNA synthesis during DNA replication, repair, and recombination. A number of DNA polymerases, such as the Taq enzyme from Thermus aquaticus, are used in various applications of molecular biology and biotechnology, in particular as DNA amplification tools. However, the efficiency of these enzymes depends on factors such as DNA origin, primer composition, template length, GC-content, and the ability to form stable secondary structures. These limitations in the use of currently known DNA polymerases lead to the search for new enzymes with improved properties. This review summarizes the main structural and molecular-kinetic features of the functioning of DNA-polymerases belonging to structural family A, including Taq polymerase. A phylogenetic analysis of these enzymes was carried out, which made it possible to establish a highly conserved consensus sequence containing 62 amino acid residues distributed over the structure of the enzyme. A comparative analysis of these amino acid residues among poorly studied DNA-polymerases revealed 7 enzymes that potentially have the properties necessary for use in DNA amplification.

[DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells].

The important role of DNA damage in the occurrence of various diseases, including cancer, has led to study of the mechanisms of genetic information stability, that have been carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and increased risk of disease development is still relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely the DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and the AP endonuclease APE1. The sensitivity of DNA probes was determined on pure enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed significant variability in the level of enzyme activity in these cell lines. These results may become a test system platform for analyzing the activity of the base excision DNA repair system in the human body.

Detection of Anthracnose in Strawberry and Methods of Etiological Diagnosis.

Anthracnose of strawberry caused by Colletotrichum fungi is a dangerous disease associated with serious damage in berry plantations. Colletotrichum nymphaeae, C. lineola, and C. godetiae have been found in Russian and international planting material of strawberry plants. The cultural and morphological characteristics are described for the isolates and nucleotide ITS1-5.8S-ITS2 sequences of fragments received are identified. It is shown that the fragments of glyceraldehyde 3-phosphate dehydrogenase and actin genes can be used to efficiently differentiate the C. lineola species from the closely related С. dematium. Two diagnostic test systems for acutatum complex identification are compared. The studied test systems do not demonstrate any false-positive results; the prepared set of С. acutatum complex-RT (ZAO Sintol) shows specificity only for the C. nymphaeae and C. fioriniae species and turned out to be nonspecific to the C. godetiae species included in the acutatum complex. The test system elaborated by Garrido et al. is found to be highly sensitive and specific to the target species of the acutatum complex.

Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity.

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg2+-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg2+ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

[Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity].

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg^(2+)-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg^(2+) ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

[Efficiency of RNA Hydrolysis by Binase from Bacillus pumilus: The Impact of Substrate Structure, Metal Ions, and Low Molecular Weight Nucleotide Compounds].

Binase is an extracellular guanyl-preferring ribonuclease from Bacillus pumilus. The main biological function of binase is RNA degradation with the formation of guanosine-2',3'-cyclic phosphate and its subsequent hydrolysis to 3'-phosphate. Extracellular RNases are believed to be key agents that affect the functional activity of the body, as they directly interact with epithelial and immune cells. The biological effects of the enzyme may consist of both direct RNA degradation, and the accumulation of 2',3'-cGMP in the human body. In this work, we have performed a comparative analysis of the cleavage efficiency of model RNA substrates, i.e., short hairpin structures that contain guanosine at various positions. It has been shown that the hydrolysis efficiency of the model RNA substrates depends on the position of guanosine. We have also demonstrated the influence of various divalent metal ions and low molecular weight nucleotide compounds on the binase-catalyzed endoribonucleolytic reaction.

The role of active-site amino acid residues in the cleavage of DNA and RNA substrates by human apurinic/apyrimidinic endonuclease APE1.

BACKGROUND: Human apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5' side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA. METHODS: Using pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition. RESULTS: Comparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3'-5'-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre-steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied. CONCLUSIONS: It was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg2+ coordination. Finally, the substitution of Met270 also destabilized the enzyme-substrate complex but did not affect the catalytic reaction. SIGNIFICANCE: It was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.

Effect of the Substrate Structure and Metal Ions on the Hydrolysis of Undamaged RNA by Human AP Endonuclease APE1.

Human apurinic/apyrimidinic (AP) endonuclease APE1 is one of the participants in the DNA base excision repair. The main biological function of APE1 is to hydrolyze the phosphodiester bond on the 5'-side of the AP sites. It has been shown recently that APE1 acts as an endoribonuclease and can cleave mRNA, thereby controlling the level of some transcripts. The sequences of CA, UA, and UG dinucleotides are the cleavage sites in RNA. In the present work, we performed a comparative analysis of the cleavage efficiency of model RNA substrates with short hairpin structures in which the loop size and the location of the pyrimidine-purine dinucleotide sequence were varied. The effect of various divalent metal ions and pH on the efficiency of the endoribonuclease reaction was analyzed. It was shown that site-specific hydrolysis of model RNA substrates depends on the spatial structure of the substrate. In addition, RNA cleavage occured in the absence of divalent metal ions, which proves that hydrolysis of DNA- and RNA substrates occurs via different catalytic mechanisms.

Search for Modified DNA Sites with the Human Methyl-CpG-Binding Enzyme MBD4.

The MBD4 enzyme initiates the process of DNA demethylation by the excision of modified DNA bases, resulting in the formation of apurinic/apyrimidinic sites. MBD4 contains a methyl-CpG-binding domain which provides the localization of the enzyme at the CpG sites, and a DNA glycosylase domain that is responsible for the catalytic activity. The aim of this work was to clarify the mechanisms of specific site recognition and formation of catalytically active complexes between model DNA substrates and the catalytic N-glycosylase domain MBD4cat. The conformational changes in MBD4cat and DNA substrates during their interaction were recorded in real time by stopped-flow detection of the fluorescence of tryptophan residues in the enzyme and fluorophores in DNA. A kinetic scheme of MBD4cat interaction with DNA was proposed, and the rate constants for the formation and decomposition of transient reaction intermediates were calculated. Using DNA substrates of different lengths, the formation of the catalytically active complex was shown to follow the primary DNA binding step which is responsible for the search and recognition of the modified base. The results reveal that in the primary complex of MBD4cat with DNA containing modified nucleotides, local melting and bending of the DNA strand occur. On the next step, when the catalytically competent conformation of the enzyme-substrate complex is formed, the modified nucleotide is everted from the double DNA helix into the active center and the void in the helix is filled by the enzyme's amino acids.

The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, Tm, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme-substrate complex is not the bottleneck controlling the catalytic activity of AAG.

Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.

Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

[STUDY OF THE EFFICACY OF REGULATION OF THE BLOOD SERUM PHYSICOCHEMICAL PARAMETERS IN CHILDREN WITH DYSFUNCTION OF PHYSIOLOGICAL SYSTEMS].

The physicochemical parameters of blood serum (osmolality, concentration of several ions, total protein, glucose) were studied in 200 children of different age with various forms of pathology. The variability of each parameter was calculated. A high level of stability of the parameters studies was revealed in healthy children and in children with dysfunction of various systems (disease of the respiratory system, gastrointestinal tract, renal and urinary tract, nervous and endocrine systems). However, estimation of their coefficients of variation showed significant individual deviations of these parameters from the average value of the examined patients. This fact reflects the extent of efficacy of activity of different organs and regulatory systems under pathological processes. Combination of clinical and ontogenetic methods of evolutionary physiology in this study opens new possibilities for understanding the nature of regulation of water-salt balance in humans and points out to the expedience of using these approaches in the practical medicine.

[Genetic ecological monitoring in human populations: heterozygosity, mtDNA haplotype variation, and genetic load].

Yu. P. Altukhov suggested that heterozygosity is an indicator of the state of the gene pool. The idea and a linked concept of genetic ecological monitoring were applied to a new dataset on mtDNA variation in East European ethnic groups. Haplotype diversity (an analog of the average heterozygosity) was shown to gradually decrease northwards. Since a similar trend is known for population density, interlinked changes were assumed for a set of parameters, which were ordered to form a causative chain: latitude increases, land productivity decreases, population density decreases, effective population size decreases, isolation of subpopulations increases, genetic drift increases, and mtDNA haplotype diversity decreases. An increase in genetic drift increases the random inbreeding rate and, consequently, the genetic load. This was confirmed by a significant correlation observed between the incidence of autosomal recessive hereditary diseases and mtDNA haplotype diversity. Based on the findings, mtDNA was assumed to provide an informative genetic system for genetic ecological monitoring; e.g., analyzing the ecology-driven changes in the gene pool.

[Incretory function of the kidney].

Participation of kidney in maintenance of constancy of parameters of the organism internal medium is connected with formation in it of physiologically active substances. They are components of systems of regulation of water-salt balance, arterial pressure, blood coagulation, erythropoiesis. Mechanisms of normalization by kidney of concentration of hormones in blood and of modulation of action of hormones are analyzed.

[Diabetes mellitus: renal osmoregulating function].

AIM: To evaluate the kidney status from osmotic urine concentration in different stages of diabetes mellitus (DM) to define whether not only glomeruli and proximal tubules, but also renal medullary substance structures are involved into the pathological process, as well as their reaction to endogenous vasopressin production. SUBJECTS AND METHODS: Forty patients with a 1-to-28-year history of DM, including 18 with diabetic nephropathy, 10 with chronic renal failure, and 22 without diabetic nephropathy, were examined. Urine and blood osmolality were determined and renal osmoregulating function was estimated. RESULTS: Decreased glomerular filtration rate was found in relation to the duration of DM. The osmolality of nocturnal urine samples tended to diminish during short-term deprivation depending on the duration of DM. Increased diuresis in DM was shown to correlate with the higher reabsorption of osmotically free water. CONCLUSION: Diminished renal concentrating capacity in DM appears to depend on evolving renal failure rather than vasopressin resistance. The application of a new approach, by calculating the clearance of sodium-free water, suggests that its increased reabsorption favors normalization of serum osmolality in hyperglycemia.

[Single-stranded DNA modification by an oligonucleotide-phthalocyanine Fe(II) conjugate: kinetic regulation and mechanism].

Catalytic oxidative modification of single-stranded DNA with hydrogen peroxide and molecular oxygen in the presence of a conjugate containing an oligonucleotide complementary to the DNA fragment and tetra-4-carboxyphthalocyanine Fe(II) was studied. The conjugate examined was found to be active in the reaction of oxidative DNA cleavage in the presence of hydrogen peroxide, like earlier studied oligonucleotide conjugates containing tetra-4-carboxyphthalocyanine Co(II) and 2,4-di-[2-(2-hydroxyethyl)]deuteroporphyrin IX Fe(III) metallocomplexes generating active oxygen forms. The new conjugate was more active in the case of oxidation with molecular oxygen. Kinetic regularities and optimal regimes of DNA oxidation with hydrogen peroxide were found.

DNA-binding and oxidative properties of cationic phthalocyanines and their dimeric complexes with anionic phthalocyanines covalently linked to oligonucleotides.

Design of chemically modified oligonucleotides for regulation of gene expression has attracted considerable attention over the past decades. One actively pursued approach involves antisense or antigene oligonucleotide constructs carrying reactive groups, many of these based on transition metal complexes. The complexes of Fe(II) and Co(II) with phthalocyanines are extremely good catalysts of oxidation of organic compounds with molecular oxygen and hydrogen peroxide. The binding of positively charged Fe(II) and Co(II) phthalocyanines with single- and double-stranded DNA was investigated. It was shown that these phthalocyanines interact with nucleic acids through an outside binding mode. The site-directed modification of single-stranded DNA by O2 and H2O2 in the presence of dimeric complexes of negatively and positively charged Fe(II) and Co(II) phthalocyanines was investigated. These complexes were formed directly on single-stranded DNA through interaction between negatively charged phthalocyanine in conjugate and positively charged phthalocyanine in solution. The resulting oppositely charged phthalocyanine complexes showed significant increase of catalytic activity compared with monomeric forms of phthalocyanines Fe(II) and Co(II). These complexes catalyzed the DNA oxidation with high efficacy and led to direct DNA strand cleavage. It was determined that oxidation of DNA by molecular oxygen catalyzed by complex of Fe(II)-phthalocyanines proceeds with higher rate than in the case of Co(II)-phthalocyanines but the latter led to a greater extent of target DNA modification.