[Correction of corneal astigmatism in femtosecond laser-assisted cataract extraction surgery]. / Korrektsiya rogovichnogo astigmatizma pri femtolazer-assistirovannoi ekstraktsii katarakty.

PURPOSE: To perform a comparative analysis of clinical and functional results and efficiency of corneal astigmatism correction after femtosecond laser-assisted cataract surgery (FLACS) with implantation of toric intraocular lens (TIOL) and in combination with arcuate keratotomy (FL-AC). MATERIAL AND METHODS: The examination included 60 patients (60 eyes). The first group consisted of 30 patients (30 eyes) who underwent FLACS with implantation of TIOL (Acrysof IQ Toric, Alcon, U.S.A.); the second group consisted of 30 patients (30 eyes) who underwent FLACS in combination with FL-AC. The examination was carried out before the operation, on the 3rd day and 3 months after surgery. Corneal astigmatism correction efficiency was analyzed using the Alpins method and graphical vector analysis. RESULTS: Uncorrected visual acuity (UCVA) indices increased to 0.70±0.24 and 0.66±0.31 in the FLACS with TIOL and FLACS with FL-AC groups, respectively, without statistically significant differences between the groups (p=0.03). The value of the residual cylinder was significantly lower in the FLACS with TIOL group (-0.77±0.58 and -0.80±0.39) compared with FLACS with FL-AC (-1.08±0.81 and -1.18±0.63) both on day 3 (p=0.02), and 3 months after surgery (p=0.02). In the FLACS with TIOL group, a higher correction index (0.99±0.18) and a smaller difference vector (0.77±0.64) were achieved compared to the FLACS with FL-AC group (0.78±0.35 and 1.05±0.76, respectively). Corneal higher-order aberrations (HOA) significantly differed on the 3rd day after surgery in the 3.0 mm (p=0.04) and 5.0 mm (p=0.04) zones between the groups. Endothelial cells density was 2460.50±328.40 and 2526.125±196.74 cl/mm2 after 3 months of observation in the FLACS with TIOL and FLACS with FL-AC groups, respectively (p=0.61). CONCLUSION: Both methods had comparable visual acuity results (p=0.03). FLACS with TIOL provided more effective correction of the cylindrical component of refraction to 3.0 D.

A kinetic mechanism of repair of DNA containing α-anomeric deoxyadenosine by human apurinic/apyrimidinic endonuclease 1.

α-Anomers of 2'-deoxyadenosine (αdA) are major products of deoxyadenosine damage when DNA is γ-irradiated under anoxic conditions. Such lesions are a threat to genomic stability and are known to be processed by human apurinic/apyrimidinic endonuclease 1 (APE1). The aim of this study was to determine whether the α-anomeric structure enhances enzyme recognition. For this purpose, we analyzed the kinetic mechanism of αdA conversion by APE1 using a stopped-flow fluorescence technique. Our data reveals that the initial formation of the complex of APE1 with an αdA-containing substrate is followed by at least three conformational transitions in this complex that correspond to the induced fit leading to the formation of a catalytically competent complex. A local perturbation around the αdA lesion in the DNA duplex allows APE1 to avoid the initial conformational changes observed earlier in the case of the enzyme binding to an undamaged ligand, abasic-site-, tetrahydrofuran-, or 5,6-dihydrouridine-containing substrates. The αdA structure promotes recognition by the enzyme but dramatically impedes formation of the catalytically competent complex and hydrolysis of the 5'-phosphodiester bond. A step following the chemical reaction, possibly a release of the αdA-containing product, is rate-limiting for the overall enzymatic process, though an α-anomeric nucleotide at the 5' terminus of the DNA nick accelerates dissociation of the enzyme-product complex. Our results show that the efficiency of αdA lesion conversion by APE1 is very low. Nonetheless, αdA repair by APE1 is probably a biologically relevant process.

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.

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.

Analysis of chromosome aberrations by FISH and Giemsa assays in lymphocytes of cancer patients undergoing whole-body irradiation: comparison of in vivo and in vitro irradiation.

PURPOSE: To study the cytogenetic effects of fractionated radiotherapy in peripheral blood lymphocytes of five cancer patients. In vitro experiments were performed in parallel using the same dose range and a comparison was made of the induced frequencies of stable and unstable chromosome aberrations. The object was to clarify the use of an in vitro calibration curve for immediate and retrospective dosimetry in cases of radiation accidents. MATERIALS AND METHODS: Patients were exposed to 60Co gamma-rays at a single dose of 11.5 cGy each day up to a total dose of 57.5 cGy, given in 5 days. For measurement of chromosome aberrations, blood was collected from patients before irradiation and after each exposure. Blood taken before treatment was used as a control and for in vitro irradiation experiments in the dose range 8-50 cGy. Chromosome aberration frequency (stable as well as unstable) was determined using fluorescence in situ hybridization (FISH) assay with specific DNA libraries for chromosomes 1, 4 and 8 and a pancentromertic probe for the whole genome. Giemsa-stained preparations were used to score unstable aberrations following in vivo and in vitro exposure. RESULTS: A linear dose-response curve was determined for both dicentrics and translocations. The in vivo frequency of translocations was higher than for dicentrics. Dose-response curves generated for translocations following in vivo and in vitro irradiation yielded similar frequencies. In contrast, for dicentrics, in vitro irradiation yielded a higher frequency when compared with data generated following in vivo exposure. CONCLUSIONS: For dose reconstruction purposes, translocations frequency seems to be a more adequate end-point than the scoring of dicentrics. The established in vitro calibration curve for dicentrics may underestimate absorbed radiation dose in cases of protracted exposure.

An investigation of the age-dependency of chromosome abnormalities in human populations exposed to low-dose ionising radiation.

Among various cytogenetic changes stable chromosome aberrations (SCHA) seem to be the most significant for ageing and carcinogenesis. Being nonlethal they can persist through cell divisions and accumulate in time. We studied the age response of SCHA (translocations and insertions) in normal and radiation exposed human populations. Two cohorts of people at the age range of 3--72 years were studied: control (43 persons) and exposed to low doses of accidental irradiation due to Chernobyl accident and atomic bomb testing in Semipalatinsk (67 persons). FISH method was used for visualisation of chromosome aberrations. Metaphases from cultured lymphocytes were hybridised with biotinilated whole chromosome specific DNA probes for 1, 4 and 12 chromosomes, and with pancentromeric probe labelled with digoxigenin. The frequency of SCHA in lymphocytes increased as a quadratic function of donor age in both populations studied, being higher in exposed cohort as compared with control one. No age dependence for dicentrics was observed. The frequency of SCHA is a reliable biomarker of ageing in humans. Quadratic model of their age-response gives reasons to suggest that their increase is due to lower level of DNA repair or/and the genomic instability in older people. The exposure of people to low doses of ionising radiation accelerates the age-related increase of SCHA frequency.

Molecular species of lecithins in human gallbladder bile.

Using a precise high performance liquid chromatography (HPLC) technique, we identified the molecular species of lecithins in gallbladder biles from patients with cholesterol gallstones (n = 29), pigment gallstones (n = 9), morbid obesity (n = 5), and "controls" (n = 10). The major lecithin species identified in all groups, in descending rank order as represented by the fatty acids in the sn-1 and sn-2 positions, were 16:0-18:2, 16:0-18:1, 16:0-20:4, 18:0-18:2, and 18:1-18:2. Lecithin species were found to be more numerous and in substantially different proportions than reported by previous investigators. No significant differences were found between any biliary lecithin species in the cholesterol and pigment stone groups. However, compared with controls, both cholesterol and pigment stone patients had smaller proportions of 16:0-20:4, the principal arachidonyl lecithin species. Using the HPLC elution sequence for quantifying the hydrophilic-hydrophobic balance, we developed a Hydrophobic Index for lecithin species in each bile based upon the principles proposed by D. M. Heuman for bile salt species. Hydrophobic indices of bile salts and lecithin were positively correlated (r = 0.48, R2 = 0.23, P = 0.0002) suggesting that more hydrophobic bile salts were associated with biliary secretion of more hydrophobic lecithins. The most hydrophobic major lecithin species, 18:0-18:2, was present in greater proportions in biles with cholesterol monohydrate crystals in their sediments and in those with cholesterol saturation indices greater than one. This work provides rigorous separation, identification, and quantitation of the lecithin species in human gallbladder bile from a large cohort of patients but, apart from a more hydrophobic bile salt pattern coupling more hydrophobic lecithins, we fail to identify any relationships of biomedical importance between lecithin species and other major biliary constituents.

Influence of total lipid concentration, bile salt:lecithin ratio, and cholesterol content on inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile.

We modified classic equilibrium dialysis methodology to correct for dialysant dilution and Donnan effects, and have systematically studied how variations in total lipid concentration, bile salt (taurocholate):lecithin (egg yolk) ratio, and cholesterol content influence inter-mixed micellar/vesicular (non-lecithin-associated) concentrations (IMC) of bile salts (BS) in model bile. To simulate large volumes of dialysant, the total volume (1 ml) of model bile was exchanged nine times during dialysis. When equilibrium was reached, dialysate BS concentrations plateaued, and initial and final BS concentrations in the dialysant were identical. After corrections for Donnan effects, IMC values were appreciably lower than final dialysate BS concentrations. Quasielastic light scattering was used to validate these IMC values by demonstrating that lipid particle sizes and mean scattered light intensities did not vary when model biles were diluted with aqueous BS solutions of the appropriate IMC. Micelles and vesicles were separated from cholesterol-supersaturated model bile, utilizing high performance gel chromatography with an eluant containing the IMC. Upon rechromatography of micelles and vesicles using an identical IMC, there was no net transfer of lipid between micelles and vesicles. To simulate dilution during gel filtration, model biles were diluted with 10 mM Na cholate, the prevailing literature eluant, resulting in net transfer of lipid between micelles and vesicles, the direction of which depended upon total lipid concentration and BS/lecithin ratio. Using the present methodology, we demonstrated that inter-mixed micellar/vesicular concentrations (IMC) values increased strongly (5 to 40 mM) with increases in both bile salt (BS):lecithin ratio and total lipid concentration, whereas variations in cholesterol content had no appreciable effects. For model biles with typical physiological biliary lipid compositions, IMC values exceeded the critical micellar concentration of the pure BS, implying that in cholesterol-supersaturated biles, simple BS micelles coexist with mixed BS/lecithin/cholesterol micelles and cholesterol/lecithin vesicles. We believe that this methodology allows the systematic evaluation of IMC values, with the ultimate aim of accurately separating micellar, vesicular, and potential other cholesterol-carrying particles from native bile.