Comparative study of treatment efficacy in severe intraepithelial squamous cell lesions and preinvasive cervical cancer by conization and chlorin e6-mediated fluorescence-assisted systemic photodynamic therapy.

BACKGROUND: Cervical cancer (CC) occupies a leading position in incidence among young women of reproductive age. In this connection, it is urgent to search for the most effective approaches to the diagnosis and treatment of this pathology. The purpose of the study was to evaluate the effectiveness of the PDT method using Cе6 with the control of the photobleaching using video and spectral fluorescence diagnostic methods, to develop the method of fluorescence-assisted systemic photodynamic therapy mediated with chlorin e6 for treatment CIN 3 and CIS. MATERIALS AND METHODS: A randomized comparative clinical study was conducted involving 94 women aged 18 to 49 years with histologically verified severe intraepithelial squamous cell lesions of the cervix or preinvasive cervical cancer. The patients were included in 2 groups: in the first group conization of the cervix was performed with curettage of the remaining part of the cervical canal; patients in the second group underwent the chlorin e6-mediated fluorescence-assisted systemic photodynamic therapy. RESULTS: The absolute majority of patients in the main group after the first course of chlorin e6-mediated fluorescence-assisted systemic photodynamic therapy showed normalization of cytological parameters and colposcopic picture, while women from the comparison group showed signs of cervical lesions statistically significantly more often. These changes corresponded to the dynamics of the proliferation markers expression in the cells of intraepithelial squamous cell lesions. Also, patients of the second group who were planning a pregnancy had better reproductive outcomes after treatment compared to those of the first group. CONCLUSION: In general, higher clinical efficacy and safety of the use of the chlorin e6-mediated fluorescence-assisted systemic photodynamic therapy in the treatment of intraepithelial squamous cell lesions and preinvasive cervical cancer have been established compared to the use of standard treatment methods.

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.

[An interaction between an obstetrician-gynecologist and a urologist in the gynecological practice].

The article is devoted to the interaction of a gynecologist and a urologist in a gynecological hospital, based on the experience of the gynecological department at the Clinic of obstetrics and gynecology of the Clinical Center of Sechenov University. The most common clinical scenarios were identified when the participation of a urologist in evaluation and treatment is urgently needed, including large pelvis mass, endometriosis with an involvement of pelvic organs, genital prolapse, small pelvis adhesions as a result of previous surgical procedures and postoperative urinary disorders. A close interaction between two specialties, which are dedicated to pelvic organs disorders, namely gynecology and urology, is extremely popular in modern clinical practice and allows to significantly reduce the number of intra- and postoperative complications, carry out a comprehensive examination and determine individual treatment tactics in a gynecological hospital, which increases quality of medical care.

A New DNA Break Repair Pathway Involving PARP3 and Base Excision Repair Proteins.

Poly(ADP-ribosyl)ation, which is catalyzed by PARP family proteins, is one of the main reactions in the cell response to genomic DNA damage. Massive impact of DNA-damaging agents (such as oxidative stress and ionizing radiation) causes numerous breaks in DNA. In this case, the development of a fast cell response, which allows the genomic DNA integrity to be retained, may be more important than the repair by more accurate but long-term restoration of the DNA structure. This is the first study to show the possibility of eliminating DNA breaks through their PARP3-dependent mono(ADP-ribosyl)ation followed by ligation and repair of the formed ribo-AP sites by the base excision repair (BER) enzyme complex. Taken together, the results of the studies on ADP-ribosylation of DNA and the data obtained in this study suggest that PARP3 may be a component of the DNA break repair system involving the BER enzyme complex.

Scope of negative solvatochromism and solvatofluorochromism of merocyanines.

A vinylogous series of highly dipolar merocyanines were designed to study their solvatochromism in a wide range of solvents including low-polarity alkanes. It has been revealed that the lower vinylogues indeed have negative solvatochromism in the full range of solvent polarities starting from n-hexane, while the hexamethinemerocyanine exhibits reversed solvatochromism. With the extreme ranges of solvatochromism, the studied dyes possess 5-7 times weaker solvatofluorochromism, which can be rationalized via their decreased dipolarity in the fluorescent state. They also demonstrate an inverse dependence of their fluorescence quantum yield on solvent polarity and have near-record Stokes shifts in high-polarity media. The experimental data are supplemented by the results of DFT quantum chemical analysis of dye electronic structures in both the ground and excited states with PCM solvent field simulation.

[Method of Surgical Management of Genital Prolapse with Cervical Elongation].

Objectives: According to different authors, the percentage of genital prolapse among gynaecological diseases that require surgical correction reaches 28−38,9%. Pelvic muscle wasting is a special kind of pelvic prolapse, often leading to cervical elongation and hypertrophy. Contemporary methods of treatment for this condition have the high rate of relapse­ 8,9−22%, thus urging to improve the existing techniques. Purpose: This research was to estimate the effectiveness of novel modification of Manchester operation in comparison with classic Manchester operation in the management of pelvic prolapse with cervical elongation. Methods: We enrolled 83 patients with pelvic prolapse and cervical elongation and divided them into two groups. In GroupI (n=47) we used the novel surgical method, supplementing original Manchester procedure with cervical stump fixation and other improvements. In GroupII we used original Manchester procedure. We compared laboratory measures as well as surgery duration, blood loss, incidence of complications, and duration of post-operational hospital stay. Patients were followed-up for 2years to estimate long-term effectiveness of surgical intervention. Statistical analysis was performed in SPSS 17.0. Results: Surgery duration in GroupII was significantly longer (47,8±26,2 vs 57,5±35,1 minutes, p<0.05). There were no significant differences in lab tests, post-operational hospital stay (5,2±0,9 vs 7,3±1,2) and incidence of post-operational complications (3 vs 4 cases). Over the 2 years of follow-up we registered 1 case of relapse in Group I and 3 cases of relapse in Group II, thus estimating the effectiveness of surgery as 97,9 vs 91,7%, a non-significant difference. We noticed that all relapsed women had signs of systemic dysplasia of connective tissue. Conclusion: Suggested modification of Manchester operation improves duration of surgical intervention itself, while providing a comparable level of effectiveness.

[Influence of calcium and rhizobial infections (Rhizobium leguminosarum) on the dynamics of nitric oxide (NO) content in roots of etiolated pea (Pisum sativum L.) seedlings].

The effect of exogenous calcium (Ca2+) and rhizobial infections (Rhizobium leguminosarum bv viceae) on the dynamics of the level of nitric oxide (NO) was studied in cross cuts of roots of two-day-old etiolated pea seedlings (Pisum sativum L.) using a DAF-2DA fluorescent probe. Fluctuations of the NO level, indicating the presence of a rhythm in the generation of NO in roots, were observed during the incubation of seedlings in water, a CaCl2 solution, and with rhizobial infections. Exogenous factors (Ca2+ and two rhizobial stamms) change the time dynamics of the NO level in comparison with the control (water).

[Influence of rhizobial (Rhizobium leguminosarum) inoculation and calcium ions on the NADPH oxidase activity in roots of etiolated pea (Pisum sativum L.) seedlings].

Changes in the functional activity of the NADPH oxidase in the microsomal fraction of roots of etiolated pea seedlings, caused by rhizobial inoculation and calcium ions (Ca2+), are shown. The enzyme activity in a medium with an exogenous source of Ca2+ (CaCl2, 100 microM) fluctuated, increasing 5 to 20 min and decreasing 10 and 30 min after addition. A calcium chelator (ethylene glycol tetraacetic acid (EDTA), 100 microM) potentiated the decrease in the enzyme activity in the presence of exogenous calcium. Rhizobial inoculation caused a 3.9-fold increase in the enzyme activity 5 min after inoculation compared to the control (without inoculation). The Ca(2+)-channel activator (amiodarone, 300 microM) and the Ca(2+)-channel blocker (lanthanum chloride, 400 microM) reduced the NADPH oxidase activity after rhizobial inoculation compared to the control level (without inoculation). It is concluded that Ca2+ and reactive oxygen species are involved in the regulation of the membrane NADPH oxidase activity in roots of pea seedlings.

[Level nitric oxide (NO) and growth of roots of etiolated pea seedlings].

Data regarding the interrelation of nitric oxide (NO) content in roots of 3-day-old etiolated pea seedlings and their growth under different concentrations of N-containing compounds were obtained. The concentration of exogenous compounds (sodium nitroprusside SNP, KNO3, NaNO2, L-arginine) rendering an inhibiting effect on the growth of roots were established, and the NO content in roots was determined at these concentration. It was shown that the inhibition of growth and highest NO content in the roots was determined with SNP (4 mM) and NaNO2 (2 mM) during 24 h exposition of seedlings. This dependence was not established in combinations with KNO3 (20 mM) and L-arginine (4 mM). We established that a NO scavenger, hemoglobin (4 µM), fully or partially removed the toxic effect of SNP, nitrate, and nitrite on growth. The effect of NO on the growth and the participation of N-containing compounds in generation of NO in roots of pea seedlings is discussed.

Kinetic mechanism of the interaction of Saccharomyces cerevisiae AP-endonuclease 1 with DNA substrates.

The apurinic/apyrimidinic endonuclease from Saccharomyces cerevisiae Apn1 is one of the key enzymes involved in base excision repair of DNA lesions. A major function of the enzyme is to cleave the upstream phosphodiester bond of an apurinic/apyrimidinic site (AP-site), leading to the formation of a single-strand break with 3'-hydroxyl (OH) and 5'-deoxyribose phosphate (dRP) termini. In this study, the pre-steady-state kinetics and conformational dynamics of DNA substrates during their interaction with Apn1 were investigated. A stopped-flow method with detection of the fluorescence intensity of 2-aminopurine and pyrrolocytosine located adjacent or opposite to the damage was used. It was found that upon interaction with Apn1, both DNA strands undergo a number of rapid changes. The location of fluorescent analogs of heterocyclic bases in DNA does not influence the catalytic step of the reaction. Comparison of data obtained for yeast Apn1 and reported data (Kanazhevskaya, L. Yu., Koval, V. V., Vorobjev, Yu. N., and Fedorova, O. S. (2012) Biochemistry, 51, 1306-1321) for human Ape1 revealed some differences in their interaction with DNA substrates.

Initiation of 8-oxoguanine base excision repair within trinucleotide tandem repeats.

Trinucleotide repeat expansion provides a molecular basis for several devastating neurodegenerative diseases. In particular, expansion of a CAG run in the human HTT gene causes Huntington's disease. One of the main reasons for triplet repeat expansion in somatic cells is base excision repair (BER), involving damaged base excision and repair DNA synthesis that may be accompanied by expansion of the repaired strand due to formation of noncanonical DNA structures. We have analyzed the kinetics of excision of a ubiquitously found oxidized purine base, 8-oxoguanine (oxoG), by DNA glycosylase OGG1 from the substrates containing a CAG run flanked by AT-rich sequences. The values of k(2) rate constant for the removal of oxoG from triplets in the middle of the run were higher than for oxoG at the flanks of the run. The value of k(3) rate constant dropped starting from the third CAG-triplet in the run and remained stable until the 3'-terminal triplet, where it decreased even more. In nuclear extracts, the profile of oxoG removal rate along the run resembled the profile of k(2) constant, suggesting that the reaction rate in the extracts is limited by base excision. The fully reconstituted BER was efficient with all substrates unless oxoG was near the 3'-flank of the run, interfering with the initiation of the repair. DNA polymerase ß was able to perform a strand-displacement DNA synthesis, which may be important for CAG run expansion initiated by BER.

Kinetic mechanism of human apurinic/apyrimidinic endonuclease action in nucleotide incision repair.

Human major apurinic/apyrimidinic endonuclease (APE1) is a multifunctional enzyme that plays a central role in DNA repair through the base excision repair (BER) pathway. Besides BER, APE1 is involved in an alternative nucleotide incision repair (NIR) pathway that bypasses glycosylases. We have analyzed the conformational dynamics and the kinetic mechanism of APE1 action in the NIR pathway. For this purpose we recorded changes in the intensity of fluorescence of 2-aminopurine located in two different positions in a substrate containing dihydrouridine (DHU) during the interaction of the substrate with the enzyme. The enzyme was found to change its conformation within the complex with substrate and also within the complex with the reaction product, and the release of the enzyme from the complex with the product seemed to be the limiting stage of the enzymatic process. The rate constants of the catalytic cleavage of DHU-containing substrates by APE1 were comparable with the appropriate rate constants for substrates containing apurinic/apyrimidinic site or tetrahydrofuran residue, which suggests that NIR is a biologically important process.

[Structural and functional characteristics of plant NADPH oxidase: a review].

Data on structural and functional characteristics of plant NADPH oxidase (Rboh) are generalized. The enzyme homologs identical to the subunit gp91(phox) of the enzymatic complex of animal cells were found in plants. The activation of Rboh depends on the influx of Ca2+ into the cytoplasm and phosphorylation of the N-terminal region of the enzyme by Ca(2+)-dependent protein kinase. The possibility of the involvement of Rop GTPase, a cytosolic component of Rboh, in the activation of Rboh is discussed. It is postulated that Rboh localizes on the plasma membrane of plant cells. Rboh is activated under the influence of both biotic and abiotic factors, which is apparently associated with Ca2+ fluxes, reactive oxygen and nitrogen species, and transduction of information to the nuclear genome.

[The NADPH oxidase activity of pea seedling roots in rhizobial infection depending on abiotic and biotic factors].

The changes in NADPH activity was studied in the roots of 3-4-day-old etiolated pea (cultivar Aksaiskii usatyi) seedlings depending on plant inoculation with Rhizobium leguminosarum bv. viceae (strain CIAM 1026), adverse environmental factors (low temperature and high dose of a mineral nitrogen fertilizer), chemical substances (sodium nitroprusside and methyl viologen, or paraquat), and a biotic factor--the bacterium Escherichia coli (strain XL-1 Blue). It was demonstrated that all exogenous factors increased the activity of microsomal NADPH oxidase. Rhizobial infection removed the activation caused by exogenous factors only in the case of high nitrogen content in the medium, thereby displaying an antagonistic effect. A synergistic action on the enzyme activity was observed in the variants with combined action of rhizobia + paraquat and rhizobia + E. coli. An increased NADPH oxidase activity coincided with a growth inhibition of pea seedling roots. The results are discussed from the standpoint of the roles of NADPH oxidase and reactive oxygen species in the legume-rhizobium symbiosis.

[Spectral and fluorescent study of the interaction of squarylium dyes, derivatives of 3H-indolium, with albumins].

Noncovalent interactions of intraionic squarylium dyes, derivatives of 3H-indolium, as well as the structurally analogous ionic indodicarbocyanine dye with serum albumins (human, bovine, rat) and, for comparison, with ovalbumin has been studied by spectral and fluorescent methods. The hydrophilic squarylium dye with sulfonate groups was found to interact with albumins more efficiently, which is probably due to the double negative charge on the dye molecule at the expense of the sulfonate groups and the ability to form hydrogen bonds with albumin. The hydrophilic indodicarbocyanine dye without the squarylium group in its structure binds to albumins much more weaker than the structurally analogous squarylium dye. The dyes bind to ovalbumin less efficiently than to serum albumins. Along with the binding of monomeric dye molecules, the aggregation of the dyes on albumins is also observed. The hydrophobic squarylium dye without sulfonate groups tends to form aggregates in aqueous solutions, which partially decompose upon the introduction of albumin into the solution. The hydrophilic squarylium dye with sulfonate groups can be recommended for tests as a spectral-fluorescent probe for serum albumins in extracellular media of living organisms.

[The influence of mineral nitrogen on legume-rhizobium symbiosis].

A literature review synthesizes the data on physiological mechanisms of the influence of high doses of mineral nitrogen (nitrates and ammonium) on the formation and functioning of legume-rhizobium symbiosis. The participation of phytohormonal and phenolic metabolism and active forms of oxygen and nitrogen in the symbiosis is highlighted. Close connection between these metabolic processes in the formation and functioning of legume-rhizobium symbiosis under a redundant supply of plants by mineral nitrogen is underlined.

Conformational dynamics of human AP endonuclease in base excision and nucleotide incision repair pathways.

APE1 is a multifunctional enzyme that plays a central role in base excision repair (BER) of DNA. APE1 is also involved in the alternative nucleotide incision repair (NIR) pathway. We present an analysis of conformational dynamics and kinetic mechanisms of the full-length APE1 and truncated NDelta61-APE1 lacking the N-terminal 61 amino acids (REF1 domain) in BER and NIR pathways. The action of both enzyme forms were described by identical kinetic schemes, containing four stages corresponding to formation of the initial enzyme-substrate complex and isomerization of this complex; when a damaged substrate was present, these stages were followed by an irreversible catalytic stage resulting in the formation of the enzyme-product complex and the equilibrium stage of product release. For the first time we showed, that upon binding AP-containing DNA, the APE1 structure underwent conformational changes before the chemical cleavage step. Under BER conditions, the REF1 domain of APE1 influenced the stability of both the enzyme-substrate and enzyme-product complexes, as well as the isomerization rate, but did not affect the rates of initial complex formation or catalysis. Under NIR conditions, the REF1 domain affected both the rate of formation and the stability of the initial complex. In comparison with the full-length protein, NDelta61-APE1 did not display a decrease in NIR activity with a dihydrouracil-containing substrate. BER conditions decrease the rate of catalysis and strongly inhibit the rate of isomerization step for the NIR substrates. Under NIR conditions AP-endonuclease activity is still very efficient.

[Interactions of pro- and eukaryotic DNA repair enzymes with oligodeoxyribonucleotides containing clustered lesions].

Interactions of oxoGuanine-DNA glycosylases from Escherichia coli (Fpg) and human (OGG1) and abasic site endonucleases from yeast (Apnl) and E. coli (Nfo) with oligodeoxyribonucleotides containing oxoGuanine (oxoG) and tetrahydrofuran (F, a stable analog of an abasic site) separated by various numbers of nucleotides have been studied. Inhibitor analysis has shown that the affinity of Fpg for single-stranded ligands does not depend on the relative positions of oxoG and F lesions. KM and kcat values have been determined for all double-stranded substrates and all enzymes under study. The effect of introducing the second lesion was strongly dependent on the relative positions of the lesions and the nature of the enzyme. The highest drop in the affinity (1.6-148-fold) and the reaction rate (4.8-58-fold) has been observed with Fpg and OGG1 for substrates containing F immediately 5' or 3' adjacent to oxoG. Introduction of the second lesion barely changed the KM values for Apnl and Nfo substrates. At the same time, the reaction rates were 5-10-fold lower for substrates containing two adjacent lesions. For all enzymes studied, increasing the distance between two lesions in duplex DNA reduced the effect of the lesion in KM and kcat values.

Electronic absorption spectra of symmetric cationic dye in constant electric field.

The electronic absorption coefficient of polymer films doped with symmetric cationic polymethine dye external electric field constant changes is researched. This effect is characterised by the short-wavelength band edge intensity increases and it decreases on the long-wavelength edge. The dye cation charge distribution in the model electric field 10(8)Vm(-1) of point charges was calculated by the method AM1. On the basis of the quantum chemical calculations the spectral regularities in electric field is interpreted by the cation electronic charge changes. The theoretical model, based on the eigenfrequencies value changes of the charged anharmonic oscillators under operation field is offered for the observed effects description. The experimental spectra well correlate with the theoretically calculated.

Structural requirements of double and single stranded DNA substrates and inhibitors, including a photoaffinity label, of Fpg protein from Escherichia coli.

Fpg protein (formamidopyrimidine or 8-oxoguanine DNA glycosylase) from E. coli catalyzes excision of several damaged purine bases, including 8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine from DNA. In this study the interaction of E. coli Fpg with various specific and nonspecific oligodeoxynucleotides was analyzed. Fpg was shown to remove 8-oxoguanine efficiently, not only from double-stranded, but also from single-stranded oligodeoxynucleotides. The Michaelis constants (KM) of a range of single-stranded oligodeoxynucleotides (0.55-1.3 microM) were shown to be 12-170 times higher that those for corresponding double-stranded oligodeoxynucleotides (KM = 6-60 nM). Depending on the position of the 8-oxoguanine within the oligodeoxynucleotides, relative initial rates of conversion of single-stranded substrates were found to be lower than, comparable to, or higher than those for double-stranded oligodeoxynucleotides. The enzyme can interact effectively not only with specific, but also with nonspecific single-stranded and double-stranded oligodeoxynucleotides, which are competitive inhibitors of the enzyme towards substrate. Fpg became irreversibly labeled after UV-irradiation in the presence of photoreactive analogs of single-stranded and double-stranded oligodeoxynucleotides. Specific and nonspecific single-stranded and double-stranded oligodeoxynucleotides essentially completely prevented the covalent binding of Fpg by the photoreactive analog. All these data argue for similar interactions occurring in the DNA binding cleft of the enzyme with both specific and nonspecific oligodeoxynucleotides. The relative affinities of Fpg for specific and nonspecific oligodeoxynucleotides differ by no more than 2 orders of magnitude. Addition of the second complementary chain increases the affinity of the first single-stranded chain by a factor of approximately 10. It is concluded that Michaelis complex formation of Fpg with DNA containing 8-oxoG cannot alone provide the major part of the enzyme specificity, which is found to lie in the kcat term for catalysis; the reaction rate being increased by 6-7 orders of magnitude by the transition from nonspecific to specific oligodeoxynucleotides.