Near-Infrared Activated Cyanine Dyes As Agents for Photothermal Therapy and Diagnosis of Tumors.

Today, it has become apparent that innovative treatment methods, including those involving simultaneous diagnosis and therapy, are particularly in demand in modern cancer medicine. The development of nanomedicine offers new ways of increasing the therapeutic index and minimizing side effects. The development of photoactivatable dyes that are effectively absorbed in the first transparency window of biological tissues (700-900 nm) and are capable of fluorescence and heat generation has led to the emergence of phototheranostics, an approach that combines the bioimaging of deep tumors and metastases and their photothermal treatment. The creation of near-infrared (NIR) light-activated agents for sensitive fluorescence bioimaging and phototherapy is a priority in phototheranostics, because the excitation of drugs and/or diagnostic substances in the near-infrared region exhibits advantages such as deep penetration into tissues and a weak baseline level of autofluorescence. In this review, we focus on NIR-excited dyes and discuss prospects for their application in photothermal therapy and the diagnosis of cancer. Particular attention is focused on the consideration of new multifunctional nanoplatforms for phototheranostics which allow one to achieve a synergistic effect in combinatorial photothermal, photodynamic, and/or chemotherapy, with simultaneous fluorescence, acoustic, and/or magnetic resonance imaging.

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[FINANCIAL AND LEGAL GUARANTEEING OF MEDICAL INSURANCE IN UKRAINE].

The objective of the research is to study the main financial and legal problems of medical insurance in Ukraine and the search for the ways of its improvement. The authors have used general scientific, special and legal methods in the process of writing the scientific article. Thus, the system method was used to study medical insurance as a type of social insurance; the structural - for studying the types of medical services provided by medical insurance; functional - to establish the subject of compensation for damage according to the results of insurance claims and to reveal its powers; comparative and legal - for the analysis of international and national theories that define the basis for organizing medical insurance and the sources of funding medical services. The authors of the article have focused on the fact that nowadays the socio-economic state of the state does not allow for providing an effective mechanism for the protection of health and medical care to the population at the expense of the funds of the existing Fund of Compulsory State Social Insurance. It has been indicated that the increase in funding for medical services and their quality within the health care is possible through the development of both voluntary and compulsory medical insurance by creating a special fund. The authors have offered to create a separate Health Insurance Fund by allocating a share of the total social contribution, which is directed to the obligatory state health insurance with the gradual transformation of this part into a separate payment of exceptionally the goal.

[CYTOKINE PROFILE IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION WITH AND WITHOUT COMPLICATIONS].

The aim of this study was to evaluate the prognostic role of the cytokine profile in patients with ST segment elevation myocardial infarction (STEMI). Materials and methods: In the present paper we studied cytokines IL-6, IL-1ß, IL-10, TNFα and CRP in 81 patients with different course of STEMI. Results: In patients with complicated course of STEMI a significant increase in the concentration of IL-6, IL-10, FNOα, CRP was recorded on the 1st, 7th and 14th days compared with control group of healthy subjects and a group of patients with uncomplicated STEMI. Concentrations of TNFα>35.49 pg/ml, IL-6>33.37 pg/ml, IL-10>34 pg/ml, CRP>10.84 mg/l on day 1 may suggest cardiovascular complications in STEMI patients within 1 year after the onset of the disease. We have not found reliable prognostic levels of IL-1ß, as the concentration of this cytokine remained within the accepted normal range.. Discussion: It is concluded that initially elevated levels of CRP l, TNFα, IL-6, IL-10 in the blood make it possible to identify groups of patients with myocardial infarction with ST-segment elevation at high risk of cardiovascular events throughout the year.

Backscattering of light from a dielectric layer on a reflecting substrate.

Scattering of light from rough dielectric films is studied experimentally. It is shown that the interference pattern of the scattered field depends critically on the power spectrum of the roughness, especially on its long-scale component. When the height of roughness is small compared with the wavelength, the backscattering peak (if it exists) is due to the interference of the singly scattered fields; hence the properties of the backscattered peak are rather unusual. In particular, the width of the peak is determined by the thickness of the film and is independent of the parameters of disorder. The intensity of the peak increases with an increase of the rms height of the surface roughness and becomes independent of the rms when the roughness is of the order of the wavelength.

Insulin-like growth factor I and II preserve myocardial structure in postinfarct swine.

BACKGROUND: Insulin-like growth factors (IGF) I and II improve myocardial function after coronary occlusion in different animal models. OBJECTIVES: To investigate the mechanism of improved myocardial function after administration of IGF-I or IGF-II in acute myocardial infarction. METHODS: Female pigs (mean (SD) weight 25 (5) kg) were subjected to acute myocardial infarction by microembolisation with 75-150 micrometer affigel blue beads. The beads contained and slowly released 150 microgram/pig of IGF-I (n = 6), IGF-II (n = 6), or pig albumin (n = 6). Echocardiography, perfusion imaging, and haemodynamic measurements were performed before infarction and during four weeks after infarction. Regional wall motion of different left ventricular segments was scored semiquantitatively on the basis of a three point scoring system, from normal = 0 to dyskinesia = 3. Serum cardiac troponin I concentration was measured before, immediately after, and three hours after the infarct. Excised hearts were analysed for actin, desmin, blood vessel density, and DNA laddering within the infarct, border, and normal myocardial areas. RESULTS: Myocardial function of the infarct related area improved significantly during the four weeks of follow up in both the IGF groups (p = 0.01). Myocardial perfusion, heart rate, and blood pressure were similar in all the animals during the study. Treated animals had lower serum cardiac troponin I concentration (p = 0.001), more actin in the border area (p = 0.01) and infarct area (p = 0.0001), and reduced DNA laddering in the infarct area compared with the controls (p < 0.05). IGF groups had more blood vessels in the border area (p = 0.04) and the infarct area (p = 0.003). CONCLUSIONS: Both types of IGF improved myocardial function and the improvement was associated with preservation of myocardial structure. IGF-I was more effective than IGF-II.

Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. Optical multichannel detection.

Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex was investigated using a novel photoactivatable dye. Laser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase. Time-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms between 325 and 650 nm. On the basis of singular value decomposition (SVD) and multiexponential fitting, three apparent lifetimes were resolved. A sequential kinetic mechanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of approximately 2.0 x 10(4) s(-1). A significant fraction of the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome c is followed by faster electron transfer from cytochrome c to Cu(A), with the equilibrium favoring the reduced cytochrome c. Subsequently, Cu(A) equilibrates with heme a with an apparent rate constant of approximately 1 x 10(4) s(-1). On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized. The extracted intermediate spectra are in excellent agreement with model spectra of the postulated intermediates, supporting the proposed mechanism.

Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome c.

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6, 8-trisulfonate in complex with cytochrome oxidase. The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6, 8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of approximately 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.

Biomaterial engineered electrodes for bioelectronics.

A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode. With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed. This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface. Biasing of the electrode at a negative potential (-0.3 V vs. SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface. Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps. While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1. The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c. The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant. At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed. Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water. Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units.

Arylazido-beta-alanine ADP-ribose, a novel irreversible competitive inhibitor of mitochondrial NADH-ubiquinone reductase.

Arylazido-beta-alanine ADP-ribose, a photoreactive analogue of ADP-ribose, was synthesized. In the dark, arylazido-beta-alanine ADP-ribose acts as a competitive reversible inhibitor of mitochondrial NADH-ubiquinone reductase with a K(i) of 37 microM. Upon photolysis, arylazido-beta-alanine ADP-ribose is converted to a potent irreversible active site-directed inhibitor of the enzyme. Photo-induced inhibition of membrane-bound NADH-ubiquinone reductase by arylazido-beta-alanine ADP-ribose is incomplete and results in a 20-fold reduction of the NADH oxidase and 2.5-fold reduction of the energy-dependent NAD(+) reductase activities. The arylazido-beta-alanine ADP-ribose resistant activities (direct and reverse) of the enzyme are characterized by a two orders of magnitude lower affinity to the corresponding substrates compared to those of the uninhibited NADH-ubiquinone reductase. A different kinetic behavior of the inhibited and native enzyme can be explained by invoking the two catalytically competent nucleotide-binding sites model of NADH-ubiquinone reductase.

Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives.

A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction.

Basic fibroblast growth factor induces myocardial hypertrophy following acute infarction in rats.

Basic fibroblast growth factor (bFGF) is a potent mitogen which induces growth of collateral vessels in ischaemic and infarcted myocardium. The effect of systemically administered bFGF on left ventricular (LV) function, myocardial hypertrophy and LV remodelling following acute myocardial infarction (MI) have not yet been fully investigated. Thirty Sprague-Dawley male rats were randomized to receive bFGF (0.5 mg) or rat albumin intraperitoneally for 1 week, beginning immediately after the induction of MI. Five animals served as controls and did not undergo any operation. Animals were killed 6 weeks after surgery and the hearts were perfused and fixed at physiological pressure. Transverse cross-sections from infarcted areas were stained with antibodies against proliferating cell nuclear antigen (PCNA) and Masson-trichrome and analysed with a coloured-image analyser for LV area (mm2), LV cavity diameter (mm), infarcted area (%), and wall thickness (mm) in infarcted and non-infarcted regions. LV area was similar in MI rats and in controls (41.7 +/- 6.9 and 43.0 +/- 1.5 mm2, respectively) and was significantly larger in MI bFGF-treated (MI/bFGF) animals (47.6 +/- 7.1 mm2) (P = 0.023). LV cavity diameter was significantly larger in the MI group than in MI/bFGF and control animals (6.0 +/- 0.8, 4.9 +/- 1.4, and 4.4 +/- 0.8 mm, respectively, P = 0.018). Wall thickness in the non-infarcted region was significantly smaller in MI animals (1.4 +/- 0.3 mm) than in MI/bFGF animals (1.6 +/- 0.4 mm) and the control group (1.6 +/- 0.1 mm) (P = 0.015). The ratio between LV cavity diameter/non-MI wall thickness was higher in MI than in control and MI/bFGF groups (4.8 +/- 1.6, 2.7 +/- 0.6 and 3.3 +/- 1.8, respectively, P = 0.03). Proliferating endothelial cells were significantly more abundant in infarcted than in normal areas in both MI and MI/bFGF groups, but with no significant differences between the groups. Intraperitoneal administration of bFGF did not cause any untoward extracardiac effects. Thus, systemic bFGF administration following acute MI in rats prevents dilatation of the LV, induces hypertrophy of the non-infarcted myocardium and exerts no untoward effects on extracardiac organs.

Comparison of energization of complex I in membrane particles from Paracoccus denitrificans and bovine heart mitochondria.

The results of preliminary studies of the effects of energization on the catalytic and EPR properties of complex I in tightly coupled membrane vesicles of Paracoccus denitrificans (SPP) are presented. They are compared to those observed in submitochondrial particles from bovine heart (SMP). All signs of energization of complex I detected by EPR in SMP (uncoupler-sensitive splitting of the gz lines of the clusters 2 and a broadening of their gxy lines, a fast-relaxing, piericidin-sensitive ubiquinone-radical signal, and a broad signal around g = 1.94) were also observed with the bacterial enzyme. There were some prominent differences, though. The signal of the fast-relaxing radicals could be evoked both in the presence or absence of reduced clusters 2, suggesting that enhancement of its spin-relaxation rate is caused by coupling to another paramagnet. The signal was hardly affected by the presence of gramicidin. The slow-relaxing radical signal did not disappear upon anaerobiosis, but was detectable for at least another 30 s. The fast-relaxing signal vanished immediately upon anaerobiosis. The activity of the bacterial enzyme during oxidation of NADH by oxygen or reduction of NAD induced by succinate oxidation, was 5-6 times higher than that of the mitochondrial enzyme. Unlike the mitochondrial enzyme, the bacterial enzyme was not inactivated by incubation at 35 degrees C. The spin concentration of the NADH-reducible [2Fe-2S] cluster (1b) was half that of the clusters 2, indicating no difference with the mitochondrial enzyme.

Continuous administration of insulin-like growth factor-I and basic fibroblast growth factor does not affect left ventricular geometry after acute myocardial infarction in rats.

We examined the long-term effect of exogenous administration of bFGF and IGF-I on myocardial geometry in 72 Sprague-Dawley male rats subjected to AMI. A preloaded miniature osmotic pump subsequently implanted in the peritoneum for continuous infusion (1 week) of IGF-I, bFGF, IGF-I+bFGF or rat albumin. Six weeks following AMI the rats were killed and cross-section slices were analyzed for left ventricular geometry. No differences were observed between IGF-I-treated, bFGF-treated, IGF-I+bFGF-treated and control groups in all parameters of the left ventricle.

Intramolecular oxidation of cytochrome c by covalently attached sulfoaromatic molecules.

Two photosensitive molecules, 1-maleimidopyrene-3,6,8-trisulfonate (MPTS) and N-acetylaminoethyl-1-aminonaphthalene-5-sulfonate (AEDANS), are employed to drive the intramolecular oxidation of the heme residue in cytochrome c. Intense pulse illumination (60-120 MW cm-2) of MPTS and AEDANS in the aqueous solution by the third harmonic frequency of Nd-Yag laser drives a two successive-photon process of the dyes. The oxidized products originating from the dyes react with variety of electron donors. MPTS and AEDANS residues were covalently linked the Saccharomyces cerevisiae iso-1-cytochrome c by labeling of its single sulfhydryl group. When pulsed by intensive laser beam the heme of the labeled ferrocytochrome c undergoes fast oxidation. Transient absorption spectroscopy was used to directly measure the rate constants for the photoinduced electron-transfer reaction from the ferros heme group to the oxidized dyes. The rate constant was found to be (3.6 +/- 0.4) x 10(4) s-1 for MPTS derivative. The rate of the heme oxidation in AEDANS derivative was faster than response time of our detection system (20 ns). Rapid photooxidation of cytochrome c makes it a useful tool for fast initiation of electron transfer in oxidized direction within complexes of cytochrome c with the other redox proteins.

The iron-sulfur clusters 2 and ubisemiquinone radicals of NADH:ubiquinone oxidoreductase are involved in energy coupling in submitochondrial particles.

The behavior of ubisemiquinone radicals and the iron-sulfur clusters 2 of NADH:ubiquinone oxidoreductase (Complex I) in coupled and uncoupled submitochondrial particles (SMP), oxidizing either NADH or succinate under steady-state conditions, was studied. Multifrequency EPR spectra revealed that the two new g2 lines of the clusters 2, only observed during coupled electron transfer under conditions where energy dissipation is rate-limiting [De Jong, A. M. Ph., Kotlyar, A. B., & Albracht, S. P. J. (1994) Biochim. Biophys. Acta 1186, 163-171], are the result of a spin-spin interaction of 2.8 mT. Investigation of the radical signals present in coupled SMP indicated that more than 90% of the radicals can be ascribed to two types of semiquinones which are bound to Complex I (QI-radicals) or ubiquinol:cytochrome c oxidoreductase (Complex III; QIII-radicals). The presence of QIII-radicals, but not that of QI-radicals, was completely abolished by uncoupler. Part of the QI-radicals weakly interact with the clusters 2 of Complex I. This uncoupler-sensitive interaction can amount to a splitting of the radical EPR signal of at most 1 mT, considerably weaker than the 2.8 mT splitting of the g2 lines of the clusters 2. We propose that the 2.8 mT splitting of these g2 lines results from an energy-induced spin-spin interaction between the two clusters 2 within the TYKY subunit of Complex I. The two clusters 2 show no interaction during electron transfer is uncoupled SMP or in fully-reduced anaerobic-coupled SMP. The results point to a direct role of the Fe-S clusters 2 and the QI-radicals in the mechanism of coupled electron transfer catalyzed by Complex I.

Use of thiouredopyrenetrisulfonate photochemistry for driving electron transfer reactions in aqueous solutions.

Photoexcitation of 1-thiouredopyrene-3,6,8-trisulfonic adducts (TUPS) of amino acids by the third harmonic frequency of a Nd:YAG laser (355 nm) generates the triplet state of the dye with high quantum efficiency. Relaxation of the triplet proceeds in anaerobiosis with a half decay time of 0.5 ms. The relaxation rate increases 100-fold in the presence of dioxygen. A radiative transition between the triplet and the ground state of the dye results in phosphorescent emission centered at 658 nm. The excited state of TUPS, being a strong reductant, can donate its electron to a variety of acceptors. Transient absorption spectroscopy was used to directly measure the photoinduced electron transfer from the excited dye to rhodamine B (RB) and cytochrome c. The reaction with RB was followed by monitoring the oxidation of the triplet state of TUPS at 487 nm (epsilon = 25 000 +/- 5 000 M-1 cm-1) or the reduction of RB at 553 nm. The second order rate constant for the reaction was found to be (2.5 +/- 0.2) x 10(9) M-1 s-1, a value compatible with that for diffusion controlled reactions. When directed to cytochrome c the photoinduced perturbation causes rapid reduction of the protein's heme group, seen as a monophasic increase of absorbance at 550 nm. The combination of appropriate redox properties with the capability of covalent protein modification makes the dye useful for initiation and analysis of electron transfer reactions in chemical and biological systems.