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Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3'-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase β-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3'-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5'-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Maltseva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

It is established that Hutchinson's ratio of the weights and sizes of the body and foraging organs in similar species is not fully obeyed in shrew species of the genus Sorex. Similarity in the centroid size of the mandible was observed in the large species S. isodon and S. araneus. Differences in the shape of the mandible, which is intensely used in foraging, were detected between the species by geometric morphometrics. Interspecific differences were found in mandibular indices, which reflect trophic specialization and functional features of the mandibles. Shrews can therefore avoid competition even when Hutchinson's rule is violated. The avoidance is due to a transformation and specialization of the mandible and changes in prey capture methods and diet, rather than to changes in size. Hutchinson's ecological rule is thus not mandatory, but is only one of the conditions for reducing competition between closely related species., (© 2023. Pleiades Publishing, Ltd.)

Different lifespans were for the first time demonstrated for three (brown, bicolor, and black) fur color morphs in ten mole vole populations of the Volga, Ural, and Trans-Ural regions. With the longest lifespan of 5 years in the species, morphs that numerically dominate in a population can live 1-4 years longer than accompanying morphs. Spearman's correlation coefficient between the longest lifespan of the morphs and their proportion in the population was R sp = 0.81 (p < 0.0001). A number of morphological and functional features were identified in the color morphs. The findings are of general biological significance, confirming the hypothesis of adaptive polymorphism. Evolutionary and ecological mechanisms whereby selective advantages develop in morphs (as probable ecomorphs) are possible to evaluate using the morphs as a natural model of the initial step of sympatric form development in different parts of the range., (© 2023. Pleiades Publishing, Ltd.)

The regulation of repair processes including base excision repair (BER) in the presence of DNA damage is implemented by a cellular signal: poly(ADP-ribosyl)ation (PARylation), which is catalysed by PARP1 and PARP2. Despite ample studies, it is far from clear how BER is regulated by PARPs and how the roles are distributed between the PARPs. Here, we investigated the effects of PARP1, PARP2 and PARylation on activities of the main BER enzymes (APE1, DNA polymerase β [Polβ] and DNA ligase IIIα [LigIIIα]) in combination with BER scaffold protein XRCC1 in the nucleosomal context. We constructed nucleosome core particles with midward- or outward-oriented damage. It was concluded that in most cases, the presence of PARP1 leads to the suppression of the activities of APE1, Polβ and to a lesser extent LigIIIα. PARylation by PARP1 attenuated this effect to various degrees depending on the enzyme. PARP2 had an influence predominantly on the last stage of BER: DNA sealing. Nonetheless, PARylation by PARP2 led to Polβ inhibition and to significant stimulation of LigIIIα activities in a NAD + -dependent manner. On the basis of the obtained and literature data, we suggest a hypothetical model of the contribution of PARP1 and PARP2 to BER.

ABSTTRACT: -Using the methods of geometric morphometrics, we have revealed the phenomenon of geographical variability of morphogenetic trajectories when doing side-by-side comparisons of mandibular shapes in individuals of different ages from three southern Trans-Ural populations of the mole vole (Ellobius talpinus Pall.) in the common morphospace.

Base excision repair (BER) ensures correction of most abundant DNA lesions in mammals. The efficiency of this multistep DNA repair process that can occur via different pathways depends on the coordinated action of enzymes catalyzing its individual steps. The scaffold XRCC1 (X-ray repair cross-complementing protein 1) protein plays an important coordinating role in the repair of damaged bases and apurinic/apyrimidinic (AP) sites via short-patch (SP) BER pathway, as well as in the repair of single-strand DNA breaks. In this study, we demonstrated for the first time in vitro formation of the ternary XRCC1 complex with the key enzymes of SP BER - DNA polymerase β (Polβ) and DNA ligase IIIα (LigIIIα) - using the fluorescence-based technique. It was found that Polβ directly interacts with LigIIIα, but their complex is less stable than the XRCC1-Polβ and XRCC1-LigIIIα complexes. The effect of XRCC1 oxidation and composition of the multiprotein complex on the efficiency of DNA synthesis and DNA ligation during DNA repair has been explored. We found that formation of the disulfide bond between Cys12 and Cys20 residues as a result of XRCC1 oxidation (previously shown to modulate the protein affinity for Polβ), affects the yield of the final product of SP BER and of non-ligated DNA intermediates (substrates of long-patch BER). The effect of XRCC1 oxidation on the final product yield depended on the presence of AP endonuclease 1. Together with the data from our previous work, the results of this study suggest an important role of XRCC1 oxidation in the fine regulation of formation of BER complexes and their functional activity.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential multifunctional protein in mammals involved in base excision DNA repair (BER), regulation of gene expression and RNA metabolism. Its major enzymatic function is incision of AP sites. Poly(ADP-ribose) polymerase 1 (PARP1) modifies itself and target proteins with poly(ADP-ribose) (PAR), contributing to regulation of many processes. To understand molecular basis of functional cooperation between APE1 and PARP1 in BER, we examined PAR-binding activity and ADP-ribosylation of human APE1 in comparison with known targets of PARP1, using the full-length, N-terminally truncated and catalytically inactive forms of APE1. The protein binds preferentially large ADP-ribose polymers, being very similar to DNA polymerase β (Polβ) but contrasting with the scaffold XRCC1 protein. The interaction with PAR involves the universally conserved catalytic portion and the eukaryote-specific extension of APE1. The ADP-ribosylation of APE1 depends on the structure of PARP1-activating DNA, contrasting APE1 with Polβ and XRCC1. Relative levels of APE1 modification in the presence of different DNA substrates were found to correlate with affinities of the DNAs for APE1 and substrate activities in the enzymatic incision, suggesting the ADP-ribosylation to occur within the DNA-mediated ternary complex. This conclusion was confirmed by importance of the length of DNA region 3' to the AP site for the modification. Deletion of the N-terminal extension of APE1 produced no significant influence on both the ADP-ribosylation efficiency and hydrolytic stability of the modified protein, suggesting localization of target amino acids in the conserved catalytic portion. The most efficient ADP-ribosylation of the catalytically inactive APE1 mutant was shown to reduce the level of PARP1 automodification, suggesting possible role of APE1 in modulating PARP1 activity. Our data on primary role of DNA in controlling the PARP-catalysed modification provide new insights into mechanisms of protein targeting for ADP-ribosylation., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The influence of XRCC1 protein oxidation on the modification of proteins catalyzed by poly(ADP-ribose)polymerases (PARP1 and PARP2) was studied for the first time. XRCC1, PARP1, and PARP2, functioning as scaffold proteins, are responsible for coordination of multistep repair of most abundant DNA lesions. We showed that the XRCC1 oxidation reduces the efficiency of its ADP-ribosylation and the protein affinity for poly(ADP-ribose). The ADP-ribose modification of various XRCC1 forms is enhanced in the presence of DNA polymerase β (Polβ), capable of forming a stable complex with XRCC1. Oxidation suppresses the inhibitory effect of XRCC1 and its complex with Polβ on the automodification of PARP1 and PARP2, which may enhance the efficiency of repair. The results of this study indicate that the oxidation of XRCC1 plays a role in fine regulation of poly(ADP-ribosyl)ation levels of proteins and their coordinating functions in DNA repair.

Base excision repair (BER) involves many enzymes acting in a coordinated fashion at the most common types of DNA damage. The coordination is facilitated by interactions between the enzymes and accessory proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1). Here we use dynamic light scattering (DLS) technique to determine the hydrodynamic sizes of several BER enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), XRCC1 and PARP1, present alone or in the equimolar mixtures with each other. From the DLS data combined with glutaraldehyde cross-linking experiments and previous quantitative binding data the oligomeric states of BER proteins and their complexes are estimated. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner. The DLS technique is applied for the first time to measure the hydrodynamic sizes of PARP1 molecules covalently bound with poly(ADP-ribose) (PAR) synthesized upon the automodification reaction. PARP1 has been detected to form huge conglomerates stabilized by Mg 2+ coordinated bonds with PAR polymers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Objective: The DNA repair enzyme tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a current inhibition target to improve the efficacy of cancer chemotherapy. Previous studies showed that compounds combining adamantane and monoterpenoid fragments are active against TDP1 enzyme. This investigation is focused on the synthesis of monoterpenoid derived esters of 1-adamantane carboxylic acid as TDP1 inhibitors., Methods: New esters were synthesized by the interaction between 1-adamantane carboxylic acid chloride and monoterpenoid alcohols. The esters were tested against TDP1 and its binding to the enzyme was modeling., Results: 13 Novel ester-based TDP1 inhibitors were synthesized with yields of 21-94%; of these, nine esters had not been previously described. A number of the esters were found to inhibit TDP1, with IC50 values ranging from 0.86-4.08 µM. Molecular modelling against the TDP1 crystal structure showed a good fit of the active esters in the catalytic pocket, explaining their potency. A non-toxic dose of ester, containing a 3,7- dimethyloctanol fragment, was found to enhance the cytotoxic effect of topotecan, a clinically used anti-cancer drug, against the human lung adenocarcinoma cell line A549., Conclusion: The esters synthesized were found to be active against TDP1 in the lower micromolar concentration range, with these findings being corroborated by molecular modeling. Simultaneous action of the ester synthesized from 3,7-dimethyloctanol-1 and topotecan revealed a synergistic effect., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Geometric morphometry has been used to reveal transformations of mandible morphogenesis in the offspring of mole voles resettled to the northern part of the species range from a southern population. The transformations were new compared to both the original (southern) and the aboriginal (northern) populations. A significant increase in the intragroup morphological disparity estimated by the mean nearest neighbor distance (MNND) in the resettled animals compared to both aboriginal populations is an indirect indication of an increased developmental instability in the resettled animals exposed to new climatic conditions.

It was found that compounds combining diazaadamantane and monoterpenoid fragments are potent inhibitors of new structural type of human recombinant DNA repair enzyme Tyrosyl-DNA phosphodiesterase I (Tdp1). It was demonstrated that the inhibition efficiency depended on the length and flexibility of the aliphatic chain of the substituent.

Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl-DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD(+). The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1-PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time., (© 2015 Authors.)

Aim: To assess changes in quality of life (QL) and cognitive functions in patients with end-stage renal failure (ESRF) treated with hemodialysis using a succinate-containing dialyzing solution (SCDS)., Subjects and Methods: Sixty-seven patients with ESRF on hemodialysis using SCDS were examined. The investigators determined QL indicators using a Russianized variant of the Kidney Disease Quality of Life Short Form (KDQOL-SF) and the indicators of cognitive functions by the Benton visual retention test intended to evaluate visual short-term memory. The 6-minute walk test was used to evaluate exercise tolerance. The patients were examined before and 6 months after initiation of SCDS use., Results: Following 6 months of SCDS treatment, the patients showed positive changes in a number of QL indicators. Their exercise capacity (p = 0.03) and the quality of sleep (p = 0.03), and social interaction (p = 0.02) were improved. The magnitude of the complaints related to disease and treatment decreased (p = 0.001). The level of patient frustration was reduced (p < 0.001). Different limitations associated with chronic kidney disease and dialysis therapy came to disturb the patients to a lesser degree (p = 0.02). The Bentoin visual retention test exhibited fewer errors (p = 0.04)., Conclusion: Hemodialysis treatment with SCDS improved a number of QL indicators and cognitive functions in the patients.

Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1-2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.

Aim: To evaluate the efficiency of using a succinate-containing dialysis solution (SCDS) in terminal renal failure patients treated with chronic hemodialysis (CHD)., Subjects and Methods: Ninety patients from two hemodialysis units took part in the crossover study and were allocated to 2 groups. For 6 months, study group patients received CHD using SCDS and control group patients had CHD with a standard bicarbonate dialysis solution after 3-month washout period followed by decussation. The time course of changes in blood biochemical parameters, 24-hour ECG monitoring data, and quality of life indicators were estimated in the patients., Results: After using acidosuccinate during hemodialysis, there was a significant reduction in the predialysis serum level of inorganic phosphate, a calcium phosphate product, gamma-glutamyl transpeptidase, urea, and aldosterone as compared to the control group. The blood concentration of total protein was also increased. After 6-month administration of acidosuccinate, the patients showed reductions in systolic blood pressure, heart rate, and the frequency and duration of ST-segment depression episodes. There were positive changes in the quality of life of patients according to the KDQOL-SF questionnaire., Conclusion: The use of SCDS in patients with CHD causes positive changes in a number of laboratory parameters and improves the physical and general status, and quality of life of patients.

Challenges of tuberculosis chemotherapy under conditions of high drug resistant (DR) tuberculosis burden are discussed. Ultimate results of treatment by standard regimens of 1658 patients with new tuberculosis cases and relapses were analyzed. Favorable ultimate results were observed among both patients with new tuberculosis cases and relapses having drugs sensitivity. Efficacy of tuberculosis treatment by standard regimens of chemotherapy is decreasing as a result of DR amplification. Risk factors of unfavorable ultimate result among pulmonary tuberculosis patients are primary resistance to isoniazid (OR = 2.1) and multiple drug resistance of M. tuberculosis (OR = 8.0). Earlier onset of treatment and correct individual therapy with second line drugs as a result of rapid methods of DR tuberculosis diagnostics are those approaches which provide the best therapeutic effect among multiple drug resistant tuberculosis patients both in culture conversion (97.7%) and cavity closure rate (82.7%).

Immunization of outbred male albino mice with live dry tularemia vaccine in a dose of 50 CFU/mouse was associated with stimulation of the NO system and accumulation of NO metabolites (nitrites and nitrates) in splenic and hepatic tissues. High levels of these metabolites persisted by day 14 after the initial and repeated immunization. These results suggest that the immunotropic effect of live dry tularemia vaccine manifested by not only modulation of the functions of immunocompetent T and B cells, NK and K cells, micro- and macrophages, but also by stimulation of intracellular anti-infection defense at the tissue level via intensification of NO synthesis.

Aim: To study specific features of the incidence, course and diagnosis of tuberculosis pericarditis (TP) in patients with HIV-infection., Material and Methods: We analysed results of diagnosis of 304 primary patients with organ tuberculosis in combination with HIV infection treated in Moscow tuberculosis hospital N 7 in 2006-2010. CD4 lymphocyte count median in tuberculosis onset was 140 in 1 mcl, 63.2% patients had a baseline level of CD4 lymphocytes under 200 in 1 mcl., Results: TP incidence in primary patients with tuberculosis and HIV-infection was 6.3% while in patients with tuberculosis of multiple locations--13.7%. Cardiac tamponade symptoms were registered only in one case. Pericardial effusion was classified as moderate and large in 68.4% patients. Patients with large effusion (more than 20 mm in isolation of pericardial leaves) have undergone diagnostic pericardiocentesis and, in some cases, microdrainage. Sensitivity of exudate test for M. tuberculosis DNA with use of polymerase chain reaction was 100%., Conclusion: Active surgical policy in massive effusion tuberculosis pericarditis in line with adequate antituberculosis and antiretrovirus therapy in HIV-infected patients results in rapid resorption of the effusion.

Aim: To analyse lethal outcomes in patients with newly-diagnosed respiratory tuberculosis comorbid with HIV-infection depending on initial count of CD4+ lymphocytes., Material and Methods: Of 304 HIV patients with newly-diagnosed tuberculosis treated in Moscow Tubercusis Hospital N 7 in 2006-2010, 40 (13.2%) patients died. Tuberculosis diagnosis was made after detection of M. tuberculosis (MT) by different tests, MT DNA in different biological material, histological verification or by effectiveness of specific antituberculous therapy. Postmortem examinations were made according to the protocol., Results: Significant differences were detected in patients with initial count of CD4+ lymphocytes less than 50 in 1 mcl. Specific CNS affection was found in patients with initial lymphocyte count CD4+ less than 100 in 1 mcl. Most of autopsy examinations registered generalized acutely progressive tuberculosis with multiple lesions of internal organs and lymph nodes (LN). Microscopy revealed obscure morphological picture of specific inflammation with prevalence of alternative-exudative tissue reactions in the absence of a productive inflammation component. Cases with submiliary dissemination which was invisible in macroscopic examination due to a bright picture of exudative tissue reaction (rare plethora of the lungs, alveolar and interstitial edema, perifocal inflammatory reaction of nonspecific reactive nature) and small size of the lesions. The comparison of clinical and autopsy diagnoses revealed that involvement of intrathoracic LN and miliary dissemination, according to autopsy, occurred much more frequently than shown by antemortem standard x-ray examination of the chest., Conclusion: It is strongly recommended to perform computed tomography of the chest in all HIV-infected patients with long-term fever but without visible alterations on chest x-ray.

The article covers analysis of regional occupational pathology service designed for primary medical and sanitary care for workers and specialized care in occupational pathology center. The authors proved cooperation between occupational pathology center and Occupational Pathology department of the Medical University to be optimal.

We studied interferon status in children during acute respiratory infections and found that it depends on etiology, course of the disease, and individual features of the organism. The efficiency of IFN inductor anaferon (pediatric formulation) and the possibility of its application in the therapy of children with acute respiratory infections were demonstrated.

The interaction of human cytoplasmic phenylalanyl-tRNA synthetase (an enzyme with yet unknown 3D-structure) with homologous tRNA(Phe) under functional conditions was studied by footprinting based on iodine cleavage of thiophosphate-substituted tRNA transcripts. Most tRNA(Phe) nucleotides recognized by the enzyme in the anticodon (G34), anticodon stem (G30-C40, A31-U39), and D-loop (G20) have effectively or moderately protected phosphates. Other important specificity elements (A35 and A36) were found to form weak nonspecific contacts. The D-stem, T-arm, and acceptor stem are also among continuous contacts of the tRNA(Phe) backbone with the enzyme, thus suggesting the presence of additional recognition elements in these regions. The data indicate that mechanisms of interaction between phenylalanyl-tRNA synthetases and specific tRNAs are different in prokaryotes and eukaryotes.

The frequency and magnitude of hepatotoxic reactions were compared in 147 new cases of pulmonary tuberculosis within the first three months of chemotherapy (CT) by standard regimen 1 [H, R, Z, S (E)] (Group 1) and regimen 2B [the same drugs + kanamycin (amikacin) and fluoroquinolones] (Group 2). Their efficiency was evaluated from 6 serum indices--the level of bilirubin, the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), and gamma-glutamyl transpeptidase (GGTP), and thymol test results. Tests were monthly carried out. The results were separately analyzed in patients with and without baseline abnormalities in the indices being tested. Within the first two months of CT, the patients without baseline abnormalities showed the slightly higher frequency and magnitude of hepatotoxic reactions on receiving regimen 2B. Following 3 months of CT combined with hepatoprotectors, the patients treated by standard regimen 1 had solitary laboratory signs of hepatic damage, but there was a regular elevation of GGTP in the regimen 2B group. After a month of regimen 1 CT in combination with hepatoprotectors, the patients with baseline abnormalities has positive changes in all the studied indices. In the patients treated by regimen 2B in combination with hepatoprotectors, the changes were the same, except for GGTP that remained to be at the increased baseline levels. Following 2 months of CT, in Group 1 positive changes continued in the studied markers and, with regimen 2B treatment, abnormal changes began increasing again. After 3 months abnormal changes were single in the markers of hepatic damage with regimen 1 treatment and there was a repeated significant rise in the values of AP and GGTP with regimen 2B. It is concluded that in addition to ALT and AST, GGTP is of great informative value in controlling the hepatotoxic effects of CT.

The immediate results of partial resections were analyzed in 120 patients with drug-resistant pulmonary tuberculosis, among whom 70 patients had multidrug resistance. A complete clinical effect (abacillation and no decay cavities) was achieved in 117 (97.5%) patients, including in 67 (95.7%) patients with multidrug resistance who showed improvement in 3 (2.5%) cases, fatal outcomes being absent.

The efficiency of treatment using the routine (I) versus IIB regime was evaluated in 161 new HIV-negative cases of destructive tuberculosis within the first 3 months of chemotherapy. The IIB regimen used in the intensive phase of chemotherapy in new tuberculosis cases before obtaining data on the drug resistance of Mycobacterium tuberculosis significantly enhance the efficiency of treatment in eliminating bacterial excretion and destructive resolution in the lung. The IIB regimen used in new tuberculosis cases with primary multidrug resistance allowed bacterial excretion to be stopped in 80% by month 3 of therapy whereas this index in the similar patients treated by the I regimen was as high as 25%. Decay cavities could be also resolved significantly more frequently by month 6 of therapy in the group of patients treated by the IIB regimen in 59% versus 29% of the patients treated by the I regimen. The inclusion of fluoroquinolones into chemotherapy caused no increase in the incidence of undesirable adverse reactions.

This review summarizes results of numerous (mainly functional) studies that have been accumulated over recent years on the problem of tRNA recognition by aminoacyl-tRNA synthetases. Development and employment of approaches that use synthetic mutant and chimeric tRNAs have demonstrated general principles underlying highly specific interaction in different systems. The specificity of interaction is determined by a certain number of nucleotides and structural elements of tRNA (constituting the set of recognition elements or specificity determinants), which are characteristic of each pair. Crystallographic structures available for many systems provide the details of the molecular basis of selective interaction. Diversity and identity of biochemical functions of the recognition elements make substantial contribution to the specificity of such interactions.

To evaluate the efficiency of chemotherapy used in patients with pulmonary tuberculosis by the results of a rapid detection of drug resistance (DR) to isoniazid and rifampicin on a "TB-Biochip" test system versus the standard treatment with its subsequent correction by the data of determination of Mycobacterium tuberculosis (MBT) resistance by the absolute concentration technique (ACT), the study included 208 patients with pulmonary tuberculosis. The patients were divided into 2 groups: 1) those in whom MBT sensitivity to antituberculous agents was determined on a "TB-Biochip" test system to detect mutations in the MBT genes rpoB, katG, inhA, ahpC that were responsible for MBT sensitivity to rifampicin and isoniazid and by ACT; 2) those in whom this was determined by only ACT. The results of a test for MBT sensitivity to rifampicin and isoniazid were obtained within 2 days before chemotherapy in Group 1 and 2 months after chemotherapy in Group 2. In Group 1, antituberculous chemotherapy was used, by taking into account MBT sensitivity to isoniazid, rifampicin, or their combination; in Group 2, the drugs were given by the standard regimens with their subsequent correction following 2 months by the results of ACT. The timely initiation of treatment with reserve drugs in the detection of drug sensitivity in MBT could achieve higher therapeutic efficiency and in a shorter space of time.

The pretreated patients who isolated drug-resistant Mycobacterium tuberculosis (MBT) were randomly divided into 2 groups. A multidrug-resistant MBT strain was isolated from 77 and 62.2% of patients in the study and control groups, respectively. Artificial pneumoperitoneum (PP) was applied to the study group patients (n = 95) during adequate chemotherapy. In the control group (n = 43), only chemotherapy was performed in accordance with the data of a MBT drug sensitivity test. The results were assessed by the trend in bacterial isolation cessation and decay cavity closure. In the group of patients treated with PP and chemotherapy, sputum inoculation abacillarity occurred in 72.6 and 95.8% by months 4 and 6, respectively; in the control group, this did in 48.8 and 65.1% in the same periods. Moreover, decay cavity closure was noted in 95.1% in the study group and in 63.7% in the control group following 6 months of treatment.

A follow-up of 558 children and adolescents contacting the patients with tuberculosis who isolated bacteria was analyzed. In 46.5% of cases, the children and adolescents contacting the patients with tuberculosis were in the foci of drug-resistant Mycobacterium tuberculosis (MBT) in the source of infection. The patients from these foci fell ill with tuberculosis 3.4 times more frequently than those from the foci where the sources of infection isolated MBT susceptible to antituberculous drugs. Processes with bacterial isolation were detected in 47.7% of the ill children and adolescents, primary MBT resistance to antituberculous drugs being noted in 68.8% of cases. Preventive chemotherapy reduces the risk for children and adolescents contacting the patients with tuberculosis to fall ill by 7.6 times. Ineffective preventive chemotherapy performed in those contacting the patients was due to the use of MBT-resistant antituberculous drugs in the source of infection. The administration of the MBT-resistant drugs in the source of infection led to the same high incidence of tuberculosis in children and adolescents from the bacterial foci of tuberculous infection as in its absence.

The effect of replacement of tRNA(Phe) recognition elements on positioning of the 3'-terminal nucleotide in the complex with phenylalanyl-tRNA synthetase (PheRS) from T. thermophilus in the absence or presence of phenylalanine and/or ATP has been studied by photoaffinity labeling with s(4)U76-substituted analogs of wild type and mutant tRNA(Phe). The double mutation G34C/A35U shows the strongest disorientation in the absence of low-molecular-weight substrates and sharply decreases the protein labeling, which suggests an initiating role of the anticodon in generation of contacts responsible for the acceptor end positioning. Efficiency of photo-crosslinking with the alpha- and beta-subunits in the presence of individual substrates is more sensitive to nucleotide replacements in the anticodon (G34 by A or A36 by C) than to changes in the general structure of tRNA(Phe) (as a result of replacement of the tertiary pair G19-C56 by U19-G56 or of U20 by A). The degree of disorders in the 3'-terminal nucleotide positioning in the presence of both substrates correlates with decrease in the turnover number of aminoacylation due to corresponding mutations. The findings suggest that specific interactions of the enzyme with the anticodon mainly promote the establishment (controlled by phenylalanine) of contacts responsible for binding of the CCA-end and terminal nucleotide in the productive complex, and the general conformation of tRNA(Phe) determines, first of all, the acceptor stem positioning (controlled by ATP). The main recognition elements of tRNA(Phe), which optimize its initial binding with PheRS, are also involved in generation of the catalytically active complex providing functional conformation of the acceptor arm.

The functional roles of phenylalanine and ATP in productive binding of the tRNA(Phe) acceptor end have been studied by photoaffinity labeling (cross-linking) of T. thermophilus phenylalanyl-tRNA synthetase (PheRS) with tRNA(Phe) analogs containing the s(4)U residue in different positions of the 3'-terminal single-stranded sequence. Human and E. coli tRNA(Phe)s used as basic structures differ by efficiency of the binding and aminoacylation with the enzyme under study. Destabilization of the complex with human tRNA(Phe) caused by replacement of three recognition elements decreases selectivity of labeling of the alpha- and beta-subunits responsible for the binding of adjacent nucleotides of the CCA-end. Phenylalanine affects the positioning of the base and ribose moieties of the 76th nucleotide, and the recorded effects do not depend on structural differences between bacterial and eukaryotic tRNA(Phe)s. Both in the absence and presence of phenylalanine, ATP more effectively inhibits the PheRS labeling with the s(4)U76-substituted analog of human tRNA(Phe) (tRNA(Phe)-s(4)U76) than with E. coli tRNA(Phe)-s(4)U76: in the first case the labeling of the alpha-subunits is inhibited more effectively; the labeling of the beta-subunits is inhibited in the first case and increased in the second case. The findings analyzed with respect to available structural data on the enzyme complexes with individual substrates suggest that the binding of phenylalanine induces a local rearrangement in the active site and directly controls positioning of the tRNA(Phe) 3'-terminal nucleotide. The effect of ATP on the acceptor end positioning is caused by global structural changes in the complex, which modulate the conformation of the acceptor arm. The rearrangement of the acceptor end induced by small substrates results in reorientation of the 3'-OH-group of the terminal ribose from the catalytic subunit onto the noncatalytic one, and this may explain the unusual stereospecificity of aminoacylation in this system.

The efficiency of treatment was compared in 2 groups of patients with destructive pulmonary tuberculosis and isolation of multidrug resistant Mycobacteria. In 43 patients of a study group, artificial pneumothorax (AP) was used during chemotherapy with reserve drugs while 43 patients of a control group received chemotherapy alone. AP was shown to be highly effective in treating patients with destructive pulmonary tuberculosis who isolated multidrug resistant Mycobacteria. Moreover, by the end of 12-month therapy, AP in combination with chemotherapy ensured cessation of bacterial isolation in 88.7% and cavernous closure in the lung in 86.8%, which was almost twice higher than that with therapy with reserve antituberculous drugs.

Two hundred and twenty-four patients with pulmonary tuberculosis and bacterial isolation were examined to study the efficiency of chemotherapy in patients who isolated drug-resistant strains of Mycobacterium tuberculosis (MBT) of different genotypes. According to the pattern of susceptibility to antituberculous drugs (ATDs), the patients were divided into 3 groups: 1) those who were found to have ATD-susceptible MBT; 2) those who had multiresistant MBT susceptible to all ATDs, other than isoniazid and rifampicin; 3) those with multidrug resistance. MBT genotyping in accordance with the polymorphism of the length of restriction fragments IS6110 has indicated that the strains of the families W and AI were prevalent in all the groups. However, the strains of mycobacteria of the W family were most frequently (62%) detected among the patients with tuberculosis with slight drug resistance. Treatment was performed in accordance with the data of tests of MBT for their susceptibility to chemical agents. Comparison of the efficiency of therapy failed to reveal any relationship of the genotype of the causative agent within the groups of patients with tuberculosis with different resistance of Mycobacteria, including those with slight drug resistance. At the same time, the pattern of drug susceptibility of MBT largely determined the frequency and trends of bacterial isolation cessation.

The frequency and pattern of adverse reactions to essential antituberculous agents were studied in 480 patients with first diagnosed and recurrent pulmonary tuberculosis. Adverse reactions were found to occur in 16.9% of cases and to be mainly associated with concomitant diseases of different organs and systems wherein they occurred 15.6 times more frequently, rather than with the number (3-4-5) of used drugs. Adverse reactions were generally caused by an individual drug and predominantly streptomycin. At the same time no significant difference was established in the frequency of toxic (45.5%) and allergic (37%) reactions, but with a rather low frequency of mixed reactions (17.3%). The firstly performed cytochemical and immunological studies of lymphocytes and eosinophils provided new evidence for the pathogenesis of different patterns of adverse reactions. Lymphocytes from patients with toxic reactions showed significant intracellular structural and metabolic disturbances that led to a higher apoptosis of these cells. In patients with lymphocytic allergic reactions, on the contrary, displayed activated processes of anaerobic oxidation and their cytotoxic activity. In both types of adverse reactions, eosinophils exhibited severe intracellular metabolic disturbances that result in destruction and increased apoptosis, which determined the allergic component in virtually all types of side effects. Rational pathogenetic therapy using hormonal, vitamin, and metabolic agents and plasmapheresis could show a 2-fold reduction in the number of patients with adverse reactions without changing the routine chemotherapy regimen and only in 5.6% of cases, the reactions were intractable, which made a specific drug be discontinued and therapy used on an individual basis.

Data concerning chemotherapy of patients with multiresistant tuberculosis of the lungs by reserve antituberculous agents in combination with ofloxacin are presented. It was shown that the ofloxacin-including chemotherapy regimen applied to patients with multiresistant destructive tuberculosis of the lungs provided by the end of the 6-month treatment course elimination of multidrug resistant tubercle bacilli isolation at least in 80% of the patients and closure of the lung caverns after artificial pneumothorax and routine surgical interventions in more than half of the patients. For all this, side effects that could not be eliminated were stated merely in 8.5% of the patients.

Aim: To study quality of life of patients with progressive renal pathology at the stage of conservative treatment., Material and Methods: Quality of life and psychological features were examined in 40 patients with renal diseases (40% males, mean age 48.6 +/- 1.3 years). 77.5% examinees suffered from glomerulonephritis, the rest had diabetic nephropathy., Results: Personality, behavioral features of the patients were characterized as well as relationships between psychological and somatic factors in development of the disease. Factors influencing quality of life of the above patients are described., Conclusion: Correction of the variables influencing, primarily, the psychological component of the quality of life and, by this component, satisfaction of the patients with their state as a whole may have a good effect on their rehabilitation.

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs., (Copyright 2002 John Wiley & Sons, Ltd.)

The present review deals with the analysis of lymphokine response in two main respiratory viral infections: influenza and respiratory syncytial (RS) infection. Immune response in these two diseases has great phenomenological differences. In particular, in RS infection the intensive response of macrophages and epithelial cells, accompanied mainly by the synthesis and secretion of tumor necrosis factor and interleukin 8, is observed. Due to this fact no protective immunity is formed in RS infection, in contrast to influenza. The specific features of immune and lymphokine response in RS infection make it difficult to develop protective vaccines. At the same time it is with RS infection that the development of chronic bronchopulmonary diseases is linked. The analysis of the role of lymphokine response in respiratory viral infections, particularly in influenza and RS infections, confirms the necessity of revising therapy both in the acute and subacute stages of these diseases.

The patients with multiresistant tuberculosis were divided into 2 groups: the sensitivity of Mycobacteria tuberculosis to antituberculous drugs was evaluated in Group 1 by the methods of absolute concentrations and in Group 2 by biological microchips determining mutations in the rpo3 gene responsible for rifampicin resistance. The results of the drug sensitivity test were obtained after 3 months of treatment in Group 1 and several days prior treatment in Group 2. By taking into account the test results, reserve drugs was used in Group 2 patients. Subsequently, the results of the drug sensitivity tests carried out by the bacteriological method in Group 2 patients showed that isoniazid resistance was simultaneously noted if there were mutations in the rpo-B gene. Timely treatment with reserve drugs exhibited higher efficiency of treatment with its shorter duration in Group 2 than in Group 1.

The efficiency of treatment was studied in 149 patients with pulmonary tuberculosis who isolated multidrug resistance of Mycobacteria tuberculosis (MBT). The multidrug resistance of MTB, to at least isoniazid and rifampicin can be associated with both the resistance to other essential (streptomycin, ethambutol) and that to reserve drugs. With this, patients with MBT resistance to a combination of essential and reserve drugs more frequently showed a chronic course of the disease with severe clinical manifestations and more disseminated infiltrative-and-destructive lesions in the lung. Drug treatment regimens using a combination of reserve drug were effective only in patients with MBT resistance to essential drugs while they were little effective in those with resistance to essential and reserve agents. The use of artificial pneumothorax in patients with MBT resistance to essential and reserve agents could cease bacterial isolation in 77.8% of the patients even by ingesting a small number of the drugs. Clinically, the occurrence of MBT resistance to reserve drugs is justified to determine a radically new status in patients in the context of chemotherapy and the whole further treatment in this group of patients. A clinical classification of MBT multidrug resistance is proposed, which identifies two categories of patients with pulmonary tuberculosis: those resistant to essential drugs and those resistant to a combination of essential and reserve drugs.

The interaction of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) with the 3;-terminal nucleotide of tRNAPhe has been studied by affinity labeling to solve the problem arising from X-ray crystallographic study: the binding sites of phenylalanine and the 3;-terminal nucleotide base were revealed to be identical in the crystal structures of PheRS complexed with the substrates. tRNAPhe derivatives containing a photoreactive 4-thiouridine (tRNAPhe-s4U-76) or 6-thioguanosine residue (tRNAPhe-s6G-76) in the 3;-end have been prepared using terminal tRNA nucleotidyl transferase. Kinetic measurements of aminoacylation provide evidence for a functional role of base-specific interactions of the 3;-terminal adenosine in productive interaction of tRNAPhe with the enzyme: tRNAPhe-s4U-76 cannot be aminoacylated; the replacement of A-76 with s6G results in a 370-fold reduction of catalytic efficiency of aminoacylation mainly due to decreased Vmax value. Relative cross-linking of the s6G-substituted tRNA to the alpha-subunit (69% of the total yield of the cross-linked alpha- and beta-subunits) is two times higher as compared to the cross-linking of tRNAPhe-s4U-76. The dialdehyde derivative, tRNAPhe-Aox-76, with periodate-oxidized 3;-terminal ribose is cross-linked with the same selectivity to the alpha-subunit as tRNAPhe-s6G-76. The results suggest specific binding of the 3;-terminal nucleotide of tRNAPhe by the catalytic subunit of PheRS in the absence of other substrates. Comparative analysis of the cross-linked products in the absence and in the presence of small substrates revealed ATP and aminoacyl-adenylate to effect the interaction of the tRNAPhe acceptor end with PheRS. The correct positioning of the 3;-terminal nucleotide of tRNAPhe corresponding to the structure of the productive complex with PheRS is therefore promoted only in the presence of all three substrates.