[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.

[Modern Approaches to Protein Engineering to Create Enzymes with New Catalytic Properties].

Adenine-DNA-glycosylase MutY is a monofunctional enzyme and catalyzes hydrolysis of N-glycosidic bonds with adenine residues located opposite 8-oxonuanine residues in DNA. Rational design was carried out to construct mutant enzyme forms with altered catalytic activity. Structures of the MutY mutants were calculated by molecular dynamics (MD). Their analysis showed that some of the MutY mutants may have AP lyase activity in addition to hydrolyzing the N-glycosidic bond, as is the case with bifunctional DNA glycosylases. MutY mutants with the A120K or S124K substitution were obtained by site-directed mutagenesis, and their catalytic activities were determined. The S120K substitution was shown to confer additional AP lyase activity, while the A124K substitution completely inactivated the enzyme.

[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.

[Initial stages of DNA Base Excision Repair in Nucleosomes].

In mammalian cells, base excision repair (BER) is the main pathway responsible for the correction of a variety of chemically modified DNA bases. DNA packaging in chromatin affects the accessibility of damaged sites to the enzymes involved in repair processes. This review presents data concerning the enzymes involved in BER. Within the nucleosome core particle (NCP), the accessibility of damaged DNA to enzymes is hindered by the presence of a histone octamer. This means that the removal of DNA lesions largely depends on their rotational and translational positioning in the NCP, as well as on the specific features of each enzyme.

[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.

[Comparative Analysis of the Activity of the Polymorphic Variants of Human Uracil-DNA-Glycosylases SMUG1 and MBD4].

The human N-glycosylases SMUG1 and MBD4 catalyze the removal of uracil residues from DNA resulting from cytosine deamination or replication errors. For polymorphic variants of SMUG1 (G90C, P240H, N244S, N248Y) and the MBD4^(cat) catalytic domain (S470L, G507S, R512W, H557D), the structures of enzyme-substrate complexes were obtained by molecular dynamic simulation. It was experimentally found that the SNP variants of SMUG1, N244S and N248Y, had increased catalytic activity compared to the wild-type enzyme, probably due to the acceleration of the dissociation of the enzyme-product complex and an increase in the enzyme turnover rate. All other SNP variants of SMUG1 (G90C, P240H) and MBD4^(cat), in which amino acid substitutions disrupted the substrate binding region and/or active site, had significantly lower catalytic activity than the wild-type enzymes.

[Priorities of national surgery. Evgeny Illarionovich Zakharov. Stomach repair with small bowel substitutes]. / Prioritety otechestvennoi khirurgii. Evgenii Illarionovich Zakharov. Tonkokishechnaya plastika zheludka.

The manuscript is devoted to the outstanding Russian surgeon Zakharov E.I.. He first performed gastrojejunoduodenoplasty in 1938 (distal stomach repair with small bowel substitute). Zakharov E.I. developed the method of post-resection stomach repair with jejunum segment interposed between the esophagus and duodenum (esophagojejunoduodenoplasty). Further studies confirmed functional advantage of gastroplasty procedures with preserved duodenal passage.

Muscle synergy for upper limb damping behavior during object transport while walking in healthy young individuals.

Transporting an object during locomotion is one of the most common activities humans perform. Previous studies have shown that continuous and predictive control of grip force, along with the inertial load force of the object, is required to complete this task successfully. Another possible CNS strategy to ensure the dynamic stability of the upper limb is to modify the apparent stiffness and damping via altered muscle activation patterns. In this study, the term damping was used to describe a reduction in upper limb vertical oscillation amplitude to maintain the orientation of the hand-held object. The goal of this study was to identify the neuromuscular strategy for controlling the upper limb during object transport while walking. Three-dimensional kinematic and surface electromyography (EMG) data were recorded from eight, right-handed, healthy young adults who were instructed to walk on a treadmill while carrying an object in their dominant/non-dominant hand, with dominant/non-dominant arm positioning but without an object, and without any object or instructed arm-positioning. EMG recordings from the dominant and non-dominant arms were decomposed separately into underlying muscle synergies using non-negative matrix factorization (NNMF). Results revealed that the dominant arm showed higher damping compared to the non-dominant arm. All muscles showed higher mean levels of activation during object transport except for posterior deltoid (PD), with activation peaks occurring around or slightly before heel contact. The muscle synergy analysis revealed an anticipatory stabilization of the shoulder and elbow joints through a proximal-to-distal muscle activation pattern. These activations appear to play an essential role in maintaining the stability of the carried object in addition to the adjustment of grip force against the perturbations caused by heel contact during walking.

Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1.

Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme's tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.

An Assay for the Activity of Base Excision Repair Enzymes in Cellular Extracts Using Fluorescent DNA Probes.

Damaged DNA bases are removed by the base excision repair (BER) mechanism. This enzymatic process begins with the action of one of DNA glycosylases, which recognize damaged DNA bases and remove them by hydrolyzing N-glycosidic bonds with the formation of apurinic/apyrimidinic (AP) sites. Apurinic/apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond on the 5'-side of the AP site with generation of the single-strand DNA break. A decrease in the functional activity of BER enzymes is associated with the increased risk of cardiovascular, neurodegenerative, and oncological diseases. In this work, we developed a fluorescence method for measuring the activity of key human DNA glycosylases and AP endonuclease in cell extracts. The efficacy of fluorescent DNA probes was tested using purified enzymes; the most efficient probes were tested in the enzymatic activity assays in the extracts of A549, MCF7, HeLa, WT-7, HEK293T, and HKC8 cells. The activity of enzymes responsible for the repair of AP sites and removal of uracil and 5,6-dihydrouracil residues was higher in cancer cell lines as compared to the normal HKC8 human kidney cell line.

[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.

Thermodynamic Analysis of Fast Stages of Specific Lesion Recognition by DNA Repair Enzymes.

The methodology of determination of the thermodynamic parameters of fast stages of recognition and cleavage of DNA substrates is described for the enzymatic processes catalyzed by DNA glycosylases Fpg and hOGG1 and AP endonuclease APE1 during base excision repair (BER) pathway. For this purpose, stopped-flow pre-steady-state kinetic analysis of tryptophan fluorescence intensity changes in proteins and fluorophores in DNA substrates was performed at various temperatures. This approach made it possible to determine the changes of standard Gibbs free energy, enthalpy, and entropy of sequential steps of DNA-substrate binding, as well as activation enthalpy and entropy for the transition complex formation of the catalytic stage. The unified features of mechanism for search and recognition of damaged DNA sites by various enzymes of the BER pathway were discovered.

Changes in proteasome chymotrypsin-like activity during the development of human mammary and thyroid carcinomas.

Changes in the proteasome chymotrypsin-like activity in mammary and thyroid carcinomas in comparison with the adjacent tissue were studied at stages T(1-4)N(0-3)M(0) and T(2-3)N(0-1)M(0), respectively. The activities changed in a wave-like manner over the course of mammary carcinoma growth in cases with and without metastases. The minimum increment of the activity in the tumor was recorded during the T(2)N(0) stage in the absence of local metastases. The increment of the activity reached the peak in N(1) tumors of the same size with metastases. The activities in the tumor and adjacent tissues virtually did not differ during the T(3-4)N(1-3) stages. The time course of proteasome activity changes in thyroid tumors of the studied stages was similar to that in mammary carcinoma. The results can be used for development of methods for evaluating the aggressiveness of mammary and thyroid tumors.

[Mechanisms of the uncomplicated wound healing in patients with ventral hernia].

84 patients operated on middle ventral hernia with the use of different methots of implantation and types of mesh implants were included in the study. All patients received the thoriugh US-investigation after the operation. The sublay hernia plasty proved to be the best reconstruction method, demonstrating lesser tissue exudation. The onlay plasty should be preserved only for cases of hernioplasty when the use of sublay technique is impossible.

[Application of roentgenendovascular embolization of the bronchial arteries in the treatment of pulmonary hemorrhage].

The results of application of roentgenendovascular enbolization of bronchial arteries in the treatment of 67 patients, suffering pulmonary hemorrhage of various etiology, are adduced. Immediate hemostatic effect was achieved in 64 (95.6%) patients. The pulmonary hemorrhage recurrence while follow-up to 1 yr have occurred in 5 (7.5%) patients. Angiographic signs of the bronchial arteries affection were adduced. The possibility of the method application in the treatment of complicated forms of malignant pulmonary and mediastinal tumors was shown.

[The catheter microenterostomy for the enteral feeding].

The main group of patients consisted of 32 patients, whom the catheter microenterostomy were used for the feeding. The control group (n=36) received the feeding through the nasointestinal double-channel transnasal combined tube. The first device proved to have minimal negative influence on the quality of life, permitting, at the same time, adequate enteral feeding, minimal frequency of lung complications and decrease of the hospital stay.

[The clinical and experimental substantiation of the laparotomic access choice].

The experimental model was constructed to choose the optimal surgical access for the treatment of postoperative ventral hernias. The experimental results were compared with the clinical study, devoted to the long-term follow-up results assessment in 508 patients. The experiment demonstrated the higher durability of the paramedian access in comparison with median and oblique assess. Its clinical use allowed to the postoperative herniation to 4.84%.

Mechanism of recognition and repair of damaged DNA by human 8-oxoguanine DNA glycosylase hOGG1.

Recent data on structural and biochemical features of human 8-oxoguanine DNA glycosylase (hOGG1) has enabled detailed evaluation of the mechanism by which the damaged DNA bases are recognized and eliminated from the chain. Pre-steady-state kinetic studies with recording of conformational transitions of the enzyme and DNA substrate significantly contribute to understanding of this mechanism. In this review we particularly focus on the interrelationship between the conformational changes of interacting molecules and kinetics of their interaction and on the nature of each elementary step during the enzymatic process. Exhaustive analysis of these data and detailed mechanism of hOGG1-catalyzed reaction are proposed.

[The role of surgical access in postoperative ventral hernia development].

Long-term follow-up of 508 patients after various surgical procedures has been conducted. Of the 351 patients were operated on using a paramedian incision, 109 had an upper median laparotomy and the rest 48 had an oblique subcostal incision. Post-operative ventral hernias were registered in 45 (8.86%) patients. The upper median laparotomy herniated in 18 (16.51%) patients, paramedian incision - in 17 (4.84%), and the oblique subcostal incision herniated in 10 (20.83%). Hernia was noticed by the patient within 12 months after the initial operation in 46.67%, of them in 85.71% - within first 6 months. 53,33% of patients were diagnosed with postoperative hernia only after thorough examination. Thus, paramedian incision is considered to be the most preferable access. Postoperative hernias develop within the first 6-12 months postoperatively, later hernia registration is a result of poor examination.

[Diagnostics and treatment of postcholecystectomy biliary complications].

The study concerns early postoperative bile leak and obstructive jaundice syndrome after cholecystectomy. 4865 patients were included in the study. The initial cholecystectomy was performed through the traditional laparotomy (n=2122), minilaparotomic access (n=1024) and laparoscopic access (n=1710). Early biliary complications were registered in 135 (2,8%) patients, of whom 47 had bile leak and 88 develop obstructive jaundice. The external drainage bile leak was registered in 0,68%; bile leak trough the trained common bile duct had 0,17% and bile leak into the abdominal cavity had 0,12% of these patients. In 17 cases the bile leak was caused by the cystic duct stump insufficiency, 12 cases were caused by bile leak from the gall bladder bed. 73,5% of bile leak were caused by misdiagnosed choledocholithiasis and papilla Vateri stenosis. Obstructive jaundice in early postoperative period was determined by underdiagnosed bile ductal pathology in the majority of patients (84 patients of 88). The main diagnostic method of biliary complications was the retrograde cholangiopancreaticography with the efficacy of 99,2%. Endoscopic transpapillary operations were curative in 97% of cases. Complications after endoscopic manipulations developed in 3,3%, all of them were successfully conservatively treated.