Spectral-fluorescent and photochemical study of 6,6'-di(benzoylamino)trimethine cyanine dyes in solutions as possible probes for DNA.

Spectral-fluorescent and photochemical properties of trimethine cyanine dyes T-304, T-306, and T-307, having substituents in 6,6'-positions, in various organic solvents, in aqueous buffer solutions, in the presence of surfactants and ethanol additives, and the effect on these properties of addition of DNA have been studied. Strong aggregation of the dyes in aqueous and aqueous buffer solutions has been shown. This is due to increased hydrophobicity of the dyes, which makes it difficult to use them as spectral-fluorescent probes for DNA. In the presence of DNA, trimethine cyanines partially form highly fluorescent complexes of dye monomers with the biomolecule, with slight decomposition of the initial aggregates and the formation of aggregates on DNA molecules. The formation of different types of dye-DNA complexes, i.e., intercalation and binding in the DNA grooves, was modeled by molecular docking. Dye-DNA complexes were also studied by circular dichroism spectroscopy and by thermal dissociation of DNA. To reveal selectivity of the dyes, their interaction with human serum albumin was briefly studied. The presence of moderate concentrations of nonionic surfactants does not lead to a significant decomposition of aggregates, but leads to a biphasic dependence of the fluorescence intensity on the DNA concentration. At the same time, ethanol additives (15%) lead to a more or less linear concentration dependence of the fluorescence intensity, which makes it possible to use these dyes as fluorescent probes for DNA. The effective binding constants of the dyes to DNA and the limits of DNA detection using the dyes in the presence of 15% ethanol were estimated. Photoisomerization and generation of the triplet states of T-304, T-306, and T-307 have been also studied. Along with the fluorescence growth, complexation with DNA leads to an increase in the yield of the triplet states of the dyes. This creates a prerequisite for using the dyes in targeted PDT. In the presence of DNA, the decay kinetics of the triplet states are biexponential, which indicates different types of dye complexes with DNA. The rate constants of oxygen quenching of the triplet states of the dyes bound to DNA are significantly lower than the diffusion-controlled values (taking into account the spin-statistical factor), which is explained by the shielding effect on the triplet molecules in complexes with DNA. The data obtained show that dyes T-304, T-306 and T-307, with addition of 15% ethanol, can be used as possible fluorescent probes for DNA.

Bilirubin in the Vitreous Body of the Eye of Human Fetuses.

The bilirubin content in the vitreous body of the eye of human fetuses of gestation week 17-31 was measured. During gestation weeks 17-19, the mean bilirubin concentration was 8.67 µmol/liter; then, decreased to 1.37 µmol/liter (gestation weeks 20 to 31). Bilirubin, being an endogenous antioxidant, is probably involved in protection of the retina and lens from LPO processes that intensively develop in retinal neurons and lens fibers.

Comparative study of the interaction of some meso-substituted anionic cyanine dyes with human serum albumin.

Spectral-fluorescent properties of the meso-substituted anionic cyanine dye 3,3'-di-(γ-sulfopropyl)-4,5,4',5'-dibenzo-9-methylthiacarbocyanine betaine (DMC) were studied in solutions and in the presence of human serum albumin (HSA). The properties of DMC were compared with those of the previously studied meso-substituted anionic dyes 3,3'-di(γ-sulfopropyl)-4,5,4',5'-dibenzo-9-ethylthiacarbocyanine betaine (DEC), 3,3'-di-(γ-sulfopropyl)-9-methylthiacarbocyanine betaine (MTC) and 3,3'-di-(γ-sulfopropyl)-5,5'-diphenyl-9-ethyloxacarbocyanine betaine (OCC), which were studied here in more detail. In aqueous solutions, DMC, like DEC, is prone to dimerization; it also forms H- and J-aggregates. The noncovalent interaction of DMC with HSA leads to decomposition of the dimers with a shift in the cis-trans isomeric equilibrium toward the trans-monomer complexed with HSA, which is accompanied by a significant increase in fluorescence. The spectral-fluorescent data were used to estimate the binding constants of the dyes with HSA and other characteristics of the dyes, which are important when used as probes for HSA. The effect of structural rearrangements of HSA upon denaturation by urea on the spectral-fluorescent properties of the dyes was studied. Molecular docking of the dye-HSA systems was performed. A comparative assessment of the prospects for the use of the dyes as spectral-fluorescent probes for HSA in vitro was carried out.

Albumin in the Vitreous Body, Retina and Lens of Human Fetal Eye.

The content of serum albumin was evaluated in the vitreous body, retina, and lens of human fetuses of 14, 16, 17, 18 and 24 weeks of gestation. Albumin was detected in these tissues. PCR analysis revealed no albumin mRNA in the retina or in the lens, while in fetal liver (control) mRNA for this protein was expressed. These findings suggest that serum albumin is not synthesized by cells of retina and lens, but is captured by them. The presence of serum albumin in human eye tissues suggests its involvement in the metabolism, maintenance of tissue volume, and antioxidant reactions.

[Effects of dexamethasone and superoxide dismutase instillations on clinical course of uveitis and local biochemical processes (experimental study)].

AIM: to evaluate and compare the effect of topical superoxide dismutase (SOD), which is an antioxidant enzyme, dexamethasone, and a combination of these on the course of experimental uveitis in rabbits as well as biochemical parameters of aqueous and vitreous humor. MATERIAL AND METHODS: Acute uveitis was induced in 16 rabbits by a double injection (subcutaneous and intravitreal) of normal horse serum. Of them 12 animals, divided into 3 groups of 4 each, received topical SOD, dexamethasone, or both daily for 7 days. The remaining 4 rabbits (8 eyes) were treated with placebo and, thus, constituted the control group. On day 8 the following parameters were measured in aqueous humor: protein concentration, antioxidant activity, SOD activity, α2-macroglobulin level, and leukocyte number. Total protein and albumin levels in vitreous humor were also determined. RESULTS: The effects of SOD and dexamethasone instillations were considered similar in many parameters. However, SOD was associated with a greater increase in antioxidant activity and a greater decrease in aqueous humor leukocytes, while dexamethasone was more effective in decreasing aqueous humor α2-macroglobulin and vitreous humor protein and albumin. The substances had a synergistic effect on iridal edema as well as aqueous humor leukocyte number and α2-macroglobulin level. CONCLUSION. Adding SOD to the complex therapy of uveitis results in lower inflammation intensity and enhanced dexamethasone effect.

[Study of the content of alpha-fetoprotein and serum albumin in the vitreous body of the eye of human embryos].

The content of serum albumin and alpha-fetoprotein in the vitreous body of the eyes of human embryos from the 16th through the 24th week was investigated. It was detected that albumin and alpha-fetoprotein in the vitreous body of human eyes are presented in equal molar concentrations in the 16th week. There is 1.5-fold increased concentration of alpha-fetoprotein in comparison to albumin during the 17th week. Seventeen weeks later, there was a reduction in the concentration of both proteins. It was reported that cyanine dye, used for detection of albumin, does not interact with alpha-fetoprotein.

α-Fetoprotein in human fetal vitreous body.

α-Fetoprotein was detected in human fetal vitreous body and its concentrations on gestation weeks 16-24 were measured. The concentration of α-fetoprotein was maximum during week 17 of pregnancy (17.4 mg/ml), but then decreased and reached 1.42 mg/ml by week 24.

[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.

[Detection of carotenoids in the vitreous body of the human eye during prenatal development].

Carotenoids were found for the first time in the vitreous body of human eye during the fetal period from week 15 until week 28. Their maximum content was timed to week 16-22. No carotenoids were found the vitreous body of 31-week fetuses, as well as adult humans, which corresponds to the published data. It was shown using HPLC that chromatographic characteristics of these carotenoids correspond to those of lutein and zeaxanthin, characteristic pigments of the retinal yellow macula.

Correlation between the content of albumin and carotenoids in human vitreous body during prenatal development.

The dynamics of the content albumin and carotenoids in human fetal vitreous body during weeks 16-31 of gestation was studied. The maximum values of total albumin (1.42 mg) and carotenoids (276 ng) during the studied period were recorded on weeks 20-22. Albumin concentration peaked during week 17 (2.11x10(-4) mol/liter) and carotenoids during weeks 16-17 (about 0.045x10(-4) mol/liter) of prenatal development. By week 31, the concentrations and total content of albumin and carotenoids in the vitreous body decreased. The physiological role of the studied components of the vitreous body for prenatal development of human eye is discussed.

Excitation energy transfer in ion pairs of polymethine cyanine dyes: efficiency and dynamics.

The present work deals with singlet excitation energy transfer (EET) occurring in contact ion pairs (CIPs) of several anionic oxonol analogues (acting as EE donors) and cationic cyanines (acting as acceptors) characterized by off resonance individual transitions. Combining conductometric and spectroscopic measurements with decreasing solvent polarity, we were able to observe a progressive ion pairing leading first to solvent-separated ion pairs (SSIPs) and then to CIPs. Analysis of the absorption spectra of three selected salts (A2,C1, A2,C2, and A1,C4) in chloroform-toluene mixtures showed that the transformation of SSIP into CIP involves the appearance of a certain exciton coupling, the extent of which decreases regularly with increasing gap between the local excitation energies. Fluorescence excitation spectra showed that EET occurs in CIP, and EET efficiencies were evaluated with a procedure expressly devised for weakly emitting donors. These were between 0.2 and 0.65 for the examined ion pairs involving anions A1 and A2. The spectroscopic study was complemented by a theoretical investigation aimed at establishing the dynamic regime of the observed EET. From classical MD simulations and local full geometry optimizations, A2,C1 and A2,C2 were found to form rather stable sandwich-type CIP structures with interchromophore distances (R) of about 0.45-0.50 nm. The donor-acceptor electronic coupling was calculated in terms of Coulombic interactions between atomic transition charges. For CIP, the electronic coupling was decidedly beyond the limit of the weak coupling required for an incoherent Förster-type mechanism. Thus, we tried to arrange the EET dynamics within the theory developed by Kimura, Kakitani, and Yamato (J. Phys. Chem. B 2000, 104, 9276) for the intermediate coupling case, which provides analytical expressions of time-dependent occupation probability, EET rate, and coherency in terms of two basic quantities: the electronic coupling and a correlation time related to the Franck-Condon factor. The latter was shown to be primarily modulated by Förster's spectral overlap integral (related in turn to the excitation energy gap). Calculations were carried out for the three sample systems using three values of the electronic coupling roughly corresponding to CIP, 1.0, and 2.0 nm interchromophore distances. At the CIP distance, EET in both A2,C1 and A2,C2 was predicted to occur with a partial exciton mechanism, very short transfer times (about 10 fs), and high degree of coherence. In A1,C4 (having the largest energy gap), EET was found to occur with a hot-transfer mechanism. More or less hot-transfer dynamics appeared to be retained by all three systems at R = 1.0 nm. Fully incoherent EET appeared to become operative only at distances larger than 2.0 nm.

[Photosensitizing properties and antioxidant activity of furagin--an antimicrobial drug that is a derivative of nitrofuran]. / Fotosensibiliziruiushchie svoistva i antioksidantnaia aktivnost' furagina--antimikrobnogo lekarstvennogo preparata, proizvodnogo nitrofurana.

Photosensitizing effect of antimicrobial drug nitrofuran derivative--furagin N-(5-nitro-2-furil)-allylidencamino-hydantoin) under irradiation with light longer than 280 nm was found. The method of investigation is based on photochemiluminescence of Gly-Trp peptide in aqueous solution. Maximum photosensitizing efficiency was observed at the furagin concentration 0.08 mM when chemiluminescence yield was 33 times greater than photochemiluminescence of Gly-Trp peptide in absence of drug. It was shown that photochemiluminescence sensitized by furagin occurred via free radical way. Life time of the triplet state of furagin determined by flash photolysis was 40 microseconds. A comparison of experimental data with kinetic calculation allowed us to estimate the rate constant of triplet quenching by oxygen ((2.2 +/- 0.3)10(8) M-1.s-1) and the total rate constants of physical quenching and chemical reaction with Gly-Trp peptide ((2.0 +/- 0.4)10(8) M-1.s-1). It was also found in experiments with photochemiluminescence of Gly-Trp peptide sensitized by riboflavin (irradiation with monochromatic light 436 nm) that furagin possesses antioxidant properties twice reducing the intensity of chemiluminescence at the drug concentration 0.029 mM.