Mechanical properties of blood exosomes and lipoproteins after the rat whole blood irradiation with X-rays in vitro explored by atomic force microscopy.

Blood is a two-component system with two levels of hierarchy: the macrosystem of blood formed elements and the dispersed system of blood nanoparticles. Biological nanoparticles are the key participants in communication between the irradiated and non-irradiated cells and inducers of the non-targeted effects of ionizing radiation. The work aimed at studying by atomic force microscopy the structural, mechanical, and electrical properties of exosomes and lipoproteins (LDL/VLDL) isolated from rat blood after its exposure to X-rays in vitro. MATERIALS AND METHODS: The whole blood of Wistar rats fed with a high-fat diet was irradiated with X-rays (1 and 100 Gy) in vitro. The structural and mechanical properties (the elastic modulus and nonspecific adhesion force) of exosome and lipoprotein isolates from the blood by ultracentrifugation method were studied using Bruker Bioscope Resolve atomic force microscope in PF QNM mode, their electric properties (the zeta-potential) was measured by electrophoretic mobility. RESULTS: Lipoproteins isolated from non-irradiated blood were softer (Me(LQ; UQ): 7.8(4.9;12.1) MPa) compared to blood nanoparticles of its exosome fraction (34.8(22.6;44.9) MPa) containing both exosomes and non-membrane nanoparticles. X-ray blood irradiation with a dose of 1 Gy significantly weakened the elastic properties of lipoproteins. Exposure of the blood to 100 Gy X-rays made lipoproteins stiffer and their nonspecific adhesive properties stronger. The radiation effects on the mechanical parameters of exosomes and non-membrane nanoparticles in exosome fractions differed. The significant radiation-induced change in electric properties of the studied nanoparticles was detected only for lipoproteins in the blood irradiated with 1 Gy X-rays. The low-dose radiation-induced changes in zeta-potential and increase in lipoprotein size with the appearance of a soft thick surface layer indicate the formation of the modified lipoproteins covered with a corona from macromolecules of irradiated blood. CONCLUSION: Our data obtained using the nanomechanical mapping mode of AFM are the first evidence of the significant radiation-induced changes in the structural and mechanical properties of the dispersed system of blood nanoparticles after the X-ray irradiation of the blood.

[The development of method of intravenous laser irradiation of blood with green laser in patients with hyperlipidemia].

The impact of intravenous laser irradiation of blood with green laser in patients with hyperlipidemia was investigated. The blood of patients was chosen as sample for analysis. The patients were divided in two groups: patients with atherosclerosis of various localization and patients with atherosclerosis associated with diabetes mellitus. The effectiveness of laser impact was evaluated according the blood biochemical indicators. The levels of crude cholesterol, triglycerides, low and very low density lipoproteins, apoproteins A and B, highly sensitive C-reactive protein, atherogenity indicator, glucose content, uric acid content were determined befor and after 1, 3 and 6 months after impact. The study results indicate the occurrence of hypolipedemic and hypoglycemic effects.

Photochemistry of bacteriochlorophylls in human blood plasma: 2. Reaction mechanism investigated by product analysis and deuterium isotope effect.

Transmetalated (Pd) bacteriochlorophyll derivatives are currently being clinically tested as sensitizers for photodynamic therapy. Protocols using short delay times between injection and irradiation generate interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the mechanism of photo-oxidation in two lipoprotein fractions, low- and high-density lipoproteins, derived from human blood plasma that preferentially accumulate these pigments (Dandler et al. [2009] Photochem. Photobiol., 85, in press). Using quenchers of reactive oxygen species, and chemical reporters, in particular peroxides generated from cholesterol as an inherent component of the lipoproteins, a Type II mechanism generating singlet oxygen has been demonstrated for Pd- and Zn-bacteriopheophorbides. In homogeneous systems, accelerated bleaching in D(2)O, compared with H(2)O, supports this mechanism. An unusual deuterium isotope effect was observed, by contrast, in heterogeneous amphiphilic-water systems. In the early phase, and under high oxygen concentrations, again a positive D-isotope effect is observed which later, in a second phase, is reversed to a negative D-isotope effect. The latter cannot be explained by heterogeneous pigment populations in the amphiphilic system; we, therefore, conclude a mechanistic switch, and discuss a possible mechanism.

Photochemistry of bacteriochlorophylls in human blood plasma: 1. Pigment stability and light-induced modifications of lipoproteins.

Transmetalated derivatives of bacteriochlorophyll are promising sensitizers in photodynamic therapy. Protocols using short delay times between injection and irradiation cause interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the photochemistry of Zn- and Pd-bacteriopheophorbide (WST09), and of the highly polar taurinated Pd-derivative, WST11, in isolated fractions of human blood plasma. The stability of all pigments is increased in blood plasma, compared with monomeric solutions. Pd-bacteriopheophorbide is much more stable than the other two derivatives. It also has a higher capacity for inducing reactive oxygen species, yet the consumption of oxygen is comparable. There is furthermore evidence for photobleaching under anoxic conditions. The generation of hydroperoxides (ROOH) is faster with Pd- than with Zn-complexes; the formation of endoperoxides (ROOR'), measured as thiobarbituric acid reactive substances, is comparable with the two central metals. Formation of both ROOH and ROOR' is increased in low-density lipoproteins (LDL) compared with high-density lipoproteins (HDL), which is probably related to the higher concentration of target molecules in the former. In HDL, extensive cross-linking is induced among the apolipoproteins; judged from the electrophoretic mobility of LDL and HDL particles, there is also a gross structural change. Photosensitized cross-linking is much less pronounced with high-density proteins.

Bridging across length scales: multi-scale ordering of supported lipid bilayers via lipoprotein self-assembly and surface patterning.

We show that a two-step process, involving spontaneous self-assembly of lipids and apolipoproteins and surface patterning, produces single, supported lipid bilayers over two discrete and independently adjustable length scales. Specifically, an aqueous phase incubation of DMPC vesicles with purified apolipoprotein A-I results in the reconstitution of high density lipoprotein (rHDL), wherein nanoscale clusters of single lipid bilayers are corralled by the protein. Adsorption of these discoidal particles to clean hydrophilic glass (or silicon) followed by direct exposure to a spatial pattern of short-wavelength UV radiation directly produces microscopic patterns of nanostructured bilayers. Alternatively, simple incubation of aqueous phase rHDL with a chemically patterned hydrophilic/hydrophobic surface produces a novel compositional pattern, caused by an increased affinity for adsorption onto hydrophilic regions relative to the surrounding hydrophobic regions. Further, by simple chemical denaturation of the boundary protein, nanoscale compartmentalization can be selectively erased, thus producing patterns of laterally fluid, lipid bilayers structured solely at the mesoscopic length scale. Since these aqueous phase microarrays of nanostructured lipid bilayers allow for membrane proteins to be embedded within single nanoscale bilayer compartments, they present a viable means of generating high-density membrane protein arrays. Such a system would permit in-depth elucidation of membrane protein structure-function relationships and the consequences of membrane compartmentalization on lipid dynamics.

Biomolecular changes and somatic mutations induced by UV laser irradiation at embryonic stage of Bombyx mori.

PURPOSE: To determine the impact of ultraviolet (UV) laser radiation on Bombyx mori embryos in terms of its effect on embryonic and larval haemolymph proteins and morphological traits. MATERIALS AND METHODS: The eggs of silkworm strain NB4D2 were exposed to third harmonic laser pulses at 355 nm from a Nd:YAG laser for different durations of 30, 60, 90, 120, 150, or 180 sec. Morphological changes induced by the UV laser were analysed at larval, pupal and adult stages. The eggs exposed to UV laser irradiation at different developmental stages were subjected to protein analysis by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). The haemolymph derived from irradiated and control larva was also analysed by SDS-PAGE. RESULTS: UV laser irradiation resulted in various structural polymorphisms. Asymmetrical fusion of segments was not confined to larva but persisted throughout pupal and adult stages. Development of extra caudal horn, unequal size and lack of antenna, retarded thoracic legs and variation in larval markings were observed. Comparatively, the effect of the UV laser on 8- and 16-h old embryo was greater than on the other stages. The changes in protein pattern were not distinct until the 5th day of embryogenesis as revealed by SDS-PAGE. A 178 kiloDalton (kDa) protein resolved into 198, 184 & 169 kDa polypeptides and 154 kDa new protein band along with other proteins of 110, 45, 41 & 38 kDa were noticed in irradiated eggs at the 6th day. Further, 33, 32, and 6.2 kDa new protein bands were observed in the haemolymph of 5th instar silkworm larvae derived from UV laser irradiated embryos. CONCLUSIONS: A comparative analysis of the present study revealed that UV laser not only induced continuous structural polymorphisms (somatic mutations), but also induced protein changes with the appearance of new protein bands in embryonic and haemolymph protein. The UV laser could be a potential tool for biochemical genetics and genome analysis in B. mori.

A comparison of the kinetics of low-density lipoprotein oxidation induced by copper or by gamma-rays: influence of radiation dose-rate and copper concentration.

The oxidation of low-density lipoproteins is the first step in the complex process leading to atherosclerosis. The aim of our study was to compare the kinetics of low density lipoprotein oxidation induced by copper ions or by oxygen free radicals generated by 60Co gamma-rays. The effects of copper concentration and irradiation dose-rate on LDL peroxidation kinetics were also studied. The oxidation of LDL was followed by the measurement of conjugated diene, hydroperoxides, and thiobarbituric acid reactive substance formation as well as alpha-tocopherol disappearance. In the case of gamma irradiation, the lag-phase before the onset of lipid peroxidation was inversely correlated to the radiation dose-rate. The radiation chemical rates (nu) increased with increasing dose-rate. Copper-induced LDL peroxidation followed two kinetic patterns: a slow kinetic for copper concentrations between 5-20 microM, and a fast kinetic for a copper concentration of 40 microM. The concentration-dependent oxidation kinetics suggest the existence of a saturable copper binding site on apo-B. When compared with gamma-rays, copper ions act as drastic and powerful oxidants only at higher concentrations (> or = 40 microM).

[Effect of incorporated ionizing radiation by cesium-137 on the state of free radical processes and the lipoprotein spectrum in rat blood]. / Vplyv inkorporovanoho ionizuiuchoho oprominennia tseziiem 137Cs na stan vil'noradykal'nykh protsesiv i sklad lipoproteïniv u krovi shchuriv.

In vivo experiments it was shown that the prolonged incorporated low levels radiation by 137Cs leads to changes in free-radical processes in blood and in lipoprotein exchange. It is considered that such changes could be very important for postradiation atherogenesis, as possible distant nonstochastic consequences of Chernobyl accident.

Effects of ionizing radiation (neutrons/gamma rays) on plasma lipids and lipoproteins in rats.

Male Wistar rats weighing 250 g were exposed to 4 Gy of neutrons/gamma radiation (3.33 Gy of neutrons and 0.66 Gy of gamma rays). After whole-body irradiation, plasma cholesterol and phospholipid levels increased up to 62 and 37%, respectively, at day 4 and then returned to control values 12 days after irradiation. Plasma triglyceride concentrations decreased concomitantly with decreased food intake after irradiation but remained higher than in pair-fed control rats. Plasma lipoproteins were separated by ultracentrifugation on a density gradient (1.006-1.210 g/ml). Four days after irradiation, most of the cholesterol (62% compared to 31% in controls, P < 0.001) is transported by apolipoprotein E-rich high-density lipoproteins. At the same time, plasma levels of apolipoproteins B and E were increased by 28 and 65%, respectively, while those of apolipoproteins AI and AIV were reduced by 21 and 59%, respectively. While in the liver of irradiated rats the apolipoprotein B/E receptor number was not modified, the hydroxymethylglutaryl coenzyme A reductase activity was fivefold higher than in control pair-fed rats. Four days after irradiation, the susceptibility of lipoproteins to peroxidation, as measured by the formation of conjugated dienes in the presence of Cu2+, was markedly increased while plasma vitamin E levels were decreased, demonstrating that irradiation reduces antioxidant stores markedly. These results suggest that such modified lipoproteins could be involved in radiation-induced vascular damage.

Alteration of lipoproteins of suction blister fluid by UV radiation.

Suction blister fluid is a good representative of the interstitial fluid feeding the epidermal cells. Lipoproteins contained in the suction blister fluid of healthy volunteers are readily photo-oxidized by UV radiation in the wavelength range 290-385 nm. Absorbed light doses equivalent to one minimal erythemal dose absorbed by skin exposed to UVB (e.g. 290-320 nm) are sufficient to induce lipid peroxidation and modification of apolipoproteins A-I, A-II and B. Albumin, which is known to protect serum fully from oxidative stresses, is not so effective against photo-oxidation. Although tryptophan (Trp) residues of proteins contained in the suction blister fluid are photo-oxidized, apolipoprotein A-II, which does not contain any chromophoric Trp residue, is also altered by the UV radiation. With regard to results obtained with a model reconstituted fluid, it is suggested that the radical chain reaction of the lipid peroxidation can transfer the initial photodamage at sites not directly susceptible to photo-oxidation. The biological implications of these results are discussed.

Artificial ultraviolet whole-body radiation does not modify serum lipoprotein, plasma fibrinogen, plasminogen or antithrombin III concentrations in post-myocardial infarction patients.

The relationship of ischaemic heart disease (IHD) to seasonal and latitude variation has prompted speculation that exposure to the ultraviolet component of solar radiation may reduce IHD risk. This hypothesis was partially tested by exposing 14 post-myocardial infarction patients to a 6 week course of artificial whole-body ultraviolet radiation (UVR). Serum lipoprotein and plasma coagulation factor concentrations were measured before and after the course of UVR. Results were compared with similar measurements from a placebo-controlled group of 13 post-myocardial patients. Despite a more than two-fold rise in mean serum 25-OHD, serum lipoprotein and plasma fibrinogen, antithrombin III and plasminogen concentrations did not change significantly in the UVR group. Significant but minor change in prothrombin time and thrombin time in the placebo group appear unlikely to be of biological significance. Seasonal and latitude variation in these IHD risk factors appear unrelated to corresponding variation in solar UVR exposure.

[The composition and physicochemical properties of the blood lipoproteins in rats exposed to external gamma irradiation]. / Sostav i nekotorye fiziko-khimicheskie svoistva lipoproteinov krovi krys pri vozdeistvii vneshnego gamma-oblucheniia.

Content of lipids, character of chemiluminescence of blood plasma and certain classes of lipoproteins have been studied. Geometrical parameters, nature and quantity of charged groups of lipoprotein particles accessible for titration have been determined 1 and 30 days after a single external gamma irradiation of rats in a dose of 3 Gy. The used irradiation dose exerts an expressed hyperlipidemic effect retained for one month after irradiation. The disturbances in the spectrum of blood lipids and lipoproteins are of hyper-beta and hyper-prebeta lipoproteinemia character. Considerable disturbances of physicochemical properties of different classes of lipoproteins have been detected. They are exhibited in changes of the pattern of free-radical processes, state of the charge of surface ionogenic groups and geometrical parameters of lipoprotein particles. Changes registered by the methods of potentiometric titration and correlation spectroscopy are most expressed in lipoproteins of very low density and those of low density.

A mechanistic study of the interaction of UVB radiations with human serum lipoproteins.

The tryptophan residues of HDL are important chromophores of the interstitial fluid feeding the epidermal cells. The UVB light readily photoionizes tryptophan residues as demonstrated by the increase in their photolysis yield in presence of N2O. Saturation of the HDL solution with a N2O/O2 mixture (80%:20%, v/v) decreases the peroxidation of HDL lipids, thereby implying that lipid peroxidation is at least partly induced by tryptophan photolysis. Addition of EDTA and desferrioxamine to HDL solutions loaded with either Cu2+ or Fe2+ ions suggests that Fe2+ could be the 'contaminating' trace metal ions that are required to explain the occurrence of lipid photoperoxidation induced by photons absorbed by tryptophan residues.

[Clinico-biochemical parallels against a background of traditional treatment and laser therapy of patients with ischemic heart disease]. / Kliniko-biokhimicheskie paralleli na fone traditsionnogo lecheniia i lazeroterapii bol'nykh ishemicheskoi bolezn'iu serdtsa.

The paper is devoted to analysis of clinico-biochemical indices and a quest for additional pathologico-chemical criteria at the cell membrane level of the efficacy of helium-neon laser therapy in coronary heart disease (CHD). Another task was to decipher a number of metabolic signs of the phenomenon of CHD "exacerbation" in laser therapy (during 4-6 sessions). Positive clinical results were obtained in 98% of the patients. A favorable time course was noted in serum lipoprotein spectrum indices and in lipid and phospholipid structure of erythrocyte membranes. Laser therapy caused mobilization of the cell membrane antioxidant defense. The 4th-6th session of irradiation was marked by temporary clinical deterioration--"exacerbation" of disease accompanied by an increase in the level of erythrocyte total phospholipids (at the expense of PTEA), a decrease in free fatty acid deficiency against a background of advancing alpha-tocopherol deficiency and a rise of the blood level of lipid peroxidation primary products (diene conjugates). A conclusion was that membrane protective drugs and drugs enhancing cell energy and antioxidant defense resources promoting the "deactivation" of influence on the membrane of lipid peroxidation metabolites (tocopherol, Essentiale, retinol, etc.) should be incorporated in therapy of CHD in order to prevent the phenomenon of its "exacerbation".