[Comparison of the effects of laser and light-emitting diodes on lipid peroxidation in rat wound exudate].

The effect of laser and light-emitting diode radiation on lipid peroxidation in rat wound exudate was studied with the aim to compare the efficiency of coherent laser and incoherent light-emitting diode radiations. A model of aseptic wound in rat suggested by L.I. Slutskii was used. A He-Ne laser (632 nm) and a U-332B light-emitting diode were used in this study. The intensity of lipid peroxidation was estimated by the TBA assay. The antioxidative capacity of rat wound fluid was evaluated by means of chemiluminescent assays in two model systems: a) aqueous system with ABAP and luminol and b) in phospholipid liposome suspension with Fe2+ and cumarin. It was shown that exposure of rat wounds to both laser and light-emitting diode radiation decreased the concentration of TBA products and increased the antioxidative capacity of wound exudates, compared with the control group (without irradiation). The results obtained show that exposure of wounds to both laser and light-emitting diode irradiation causes a decrease in the oxidative stress in the rat wound fluid. No significant quantitative difference between the effects of laser and light-emitting diode irradiation was found.

[A comparison of the effects of laser and light-emitting diodes on superoxide dismutase activity and nitric oxide production in rat wound fluid].

The action of laser and light-emitting diode radiation in the visible region on the content of reactive nitrogen species and activity of superoxide dismutase in rat wound fluid was studied, and efficiency of action of coherent laser and incoherent light emitting diode radiations in the red region of the spectrum on the parameters under study was compared. A model of incised aseptic wounds in rats proposed by L.I. Slutskiy was used. A He-Ne laser (632 nm) and a Y-332B light emitting diode served as radiation sources. It was shown that (1) exposure of wounds to the visible light of both laser and light-emitting diodes causes dose-dependent changes in superoxide dismutase activity and production of nitrites and (2) the radiation coherence does not play any significant role in the changes of superoxide dismutase activity or nitrogen oxide formation by wound fluid phagocytes.

[The role of lipid peroxidation products in the effect of He-Ne laser on human blood leukocytes].

The role of lipid peroxidation products formed in membranes of human blood leukocytes after irradiation with He-Ne laser was studied. It was found that low-intensity laser irradiation (0.3-1.6 J/cm2) leads to both cell activation and an increase in the content of lipid peroxidation products. The intensity of lipid peroxidation was analyzed by estimating the amount of TBA reactive products and lipid diene conjugates. Irradiation in the presence of an exogenous photosensitizer (protoporphyrin IX) enhanced the phenomena observed. The use of antioxidants (tocopherol and ionol) completely eliminated the laser-induced effects (changes in leukocyte activity and accumulation of lipid peroxidation products). These results can be explained by the fact that laser irradiation leads to the activation of lipid peroxidation in leukocyte membranes, which in turn enhances the response of cells to the stimulus (priming).

[The role of blood plasma porphyrins in the effects of He-Ne-laser on human leukocytes].

The He-Ne-laser induced effects in human blood leukocytes in the presence of autologic plasma were investigated. Experiments were performed in two ways: (1) He-Ne-laser irradiation of cells in the presence of autologic plasma or (2) laser irradiation and subsequent addition of autologic plasma to the cell suspension. The concentration dependencies of plasma additions were evaluated. To obtain different concentrations of porphyrins in plasma samples, we either diluted the samples with PBS or selected patients with different porphyrin plasma content. The effects of He-Ne-laser irradiation were characterized by the maximum effect dose (Dmax) of irradiation and the degree of maximum cell activation (Amax, priming index). In the first series of experiments, we irradiated leukocytes in the presence of autologic plasma taken from patients with pneumonia and bronchial asthma. It was found that Dmax decreased with increasing porphyrin concentration in plasma. It was observed that, at low porphyrin concentrations, Amax increased severalfold with increasing photosensitizer concentration. At a porphyrin concentration of 0.46 pmol a decrease in Amax was detected as the porhyrin concentration increased. The same effects were revealed at high doses of laser irradiation. Very similar effects were found in experiments with the addition of irradiated plasma to cells. However, the Amax value was considerably less compared to that after irradiation in the presence of plasma (160% vs. 230 - 270% upon combined irradiation of cells and plasma). The Dmax value was higher in the series of experiments in which plasma was irradiated separately. The results suggest that laser-induced leukocyte activation can be mediated by blood plasma porphyrins and the products of lipid peroxidation formed as a result of porphyrin-photosensitized lipid oxidation.

[A comparative study of the effects of laser and light-emitting diode irradiation on the wound healing and functional activity of wound exudate leukocytes].

The effects of coherent He-Ne laser and non-coherent light-emitting diode radiation on rat skin wound healing and functional activity of wound excudate leukocytes were compared. A comparative pathomorphological analysis showed that the He-Ne laser and light-emitting diode irradiation stimulated the transition of the inflammatory phase of the wound healing into the reparative (proliferative) and scarring phases sequentially. It was also detected that the functional activity of leucocytes changed in a dose-dependent manner. The leukocyte activity was found to be similar in the groups with laser and light-emitting diode irradiation. Thus, we can conclude that coherent laser and non-coherent light-emitting diode radiation have very close effects on wound healing and activity of wound exudate leukocytes, and coherence is not required for this activity.

Low-power laser irradiation induces leukocyte priming.

Laser radiation (LR) of various spectral composition has been broadly used in clinical practice. However, the mechanism of the stimulating effects of LR remains obscure. The effect of He-Ne LR (633 nm) on human blood leukocytes was investigated both in the absence and presence of 8.65 nmol/l phthalocyanine (PhC). Irradiation of non-stimulated leukocytes with 0.025 to 0.5 J/cm2 did not lead to any activation of their luminol-dependent chemiluminescence (LCL). On the other hand, LR increased in most cases the subsequent CL response of the cells to opsonized zymosan (priming action of He-Ne-laser light). The effect of LR on the leukocytes was not standard. In irradiated leukocytes isolated from patients with severe acute or chronic pneumonia, or chronic bronchitis, the maximal LCL exceeded that for non-irradiated cells by 80% (0.05 J/cm2), 20-25% (0.15 J/cm2), and 0%, respectively (doses are shown in parentheses). Further increase of the exposure brought about a dose-dependent inhibition of LCL in cells from patients with severe acute and chronic pneumonia. There was an intriguing relationship between maximal CL responses of leukocytes subjected to laser irradiation in the presence and without PhC. When the priming effect of LR on isolated cells was small, it increased in the presence of exogenous photosensitizer, phthalocyanine; in cells of severely ill patients where the initial effect of LR was strong, Pc inhibited the priming action of LR. Apparently, different cells contained different amounts of endogenous photosensitizer(s); the addition of exogenous sensitizer increased the priming action of LR at low concentrations and decreased it at higher concentrations of the endogenous photosensitizer.

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes.

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.

[Fluorescent probe study of temperature-dependent conformational transitions in proteins]. / Izuchenie temperaturno-zavisimykh konformatsionnykh perekhodov belkov metodom fluorestsentnykh zondov

Temperature-dependent conformational features of human serum (SAH) and egg albumins were studied by fluorescent probes such as 3-metoxybenzantron (MBA), dimethylaminocholkon (DMC) and 1-anilononaphtalene-8-sulfonate (ANS) in temperature range of 4--68 degrees C. It was found that these proteins undergo the conformational transitions accompanied by the changes of the quantrum yield and maximum position of fluorescence of MBA, DMA and ANS at 12--16 degrees C, 16--23 degrees C, 40--62 degrees C for SAH and at 5--25 degrees C, 46--64 degrees C for egg albumin.

[Inhibition of the antioxidative activity of plasma by sodium azide]. / Ingibirovanie antiokislitel'noi aktivnosti plazmy krovi azidom natriia.

Powerful antioxidant activity of human plasma was demonstrated by measuring the thiobarbituric acid reaction and Fe+2-induced chemiluminescence. Inhibition of lipid peroxidation was shown both for plasma lipids and for the suspension of egg lipoproteins, which was taken as a model system. The inhibitory effect of plasma peroxidation was removed by azide Na taken in the concentration of 0.5 mg/ml, but caeroplasmin activity in the plasma was completely suppressed at NaN3 concentration equal to 0.1 mg/ml. A low correlation (r = 0.75) between caeruloplasmin activity in the blood plasma and extent of chemiluminescence activation obtained in the presence of NaN3 was found. The presented data led to an assumption that only a part of lipid peroxidation inhibitors in the plasma can be attributed with caeruloplasmin.

[Effect of low intensity laser light in the red range on macrophage superoxide dismutase activity]. / Vliianie nizkointensivnogo lazernogo izlucheniia krasnogo diapazona na aktivnost' superoksiddismutazy makrofagov.

The effect of lipopolysaccharides (LPS) and low-power laser radiation on nitric oxide (NO) production and the activity of superoxide dismutase (SOD) in rat peritoneal exudate macrophages was investigated. It was found that LPS increased NO production and SOD activity in macrophages in a concentration-dependent manner. The maximal activity of SOD was more than 100 times greater than in the control and was achieved at an LPS concentration of less than 10 ng/ml. The maximal production of NO was more than 250% of the control and was achieved at an LPS concentration of 100 ng/ml. The presence of cycloheximide, a transcriptional synthesis inhibitor, in conditioned media almost completely abolished SOD activation and NO production. This implies that the LPS-induced increase in SOD activity and NO production were determined by the activation of de novo protein synthesis (SOD and inducible NO synthase). The irradiation of macrophages by red light resulted in a dose-dependent increase in NO production and SOD activity. The incubation of irradiated cells in the presence of 10 microM cycloheximide abolished the increase. The presence of antioxidants (mexidol and ascorbate) also significantly inhibited the laser-induced activity of macrophages. Thus, laser irradiation of cells in the red range activates the synthesis of SOD and inducible NO-synthase de novo due to photosensitized initiation of free radical reactions.

[Phase transitions of model and biological membranes. II. Temperature-dependent structural reorganization of sarcoplasmic reticulum membranes]. / Izuchenie fazovykh perekhodov model'nykh i biologicheskikh membran. II. Temperaturno-zavisimye strukturnye perestroiki membran sarkoplazmaticheskogo retikuluma.

Temperature-dependent structural transitions of sarcoplasmic reticulum membranes, Ca2+-activated ATPase from membranes and liposomes formed from total membrane lipids were studied by fluorescent probes such as 3-metoxybenzantron and dimetylaminochalkon. It was found that sarcoplasmic reticulum membranes undergo the structural transitions at 10, 17--18, 30, 41--43 and 49 degrees C; lipids at 14--15, 30 degrees C and ATPase at 47--49 degrees C. The data obtained here indicate that transitions at 10, 17--18 and 30 degrees C in sarcoplasmic reticulum membranes are associated with a melt of lipids, while the transitions at 41--43 and 49 degrees C apparently reflect changes in conformation of membrane proteins.

[Registration of surface fluorescence of endogenous porphyrins during low-level laser therapy of gastric and duodenal ulcers]. / Izmerenie poverkhnostei fluorestsentsii éndogennykh porfirinov v protsesse lazeroterapii iazv zheludka i dvenadtsetiperstnoi kishki.

With the use of the developed opto-fiber spectroscope and gastroscope, the intensity of fluorescence from the ulcer surface into the gastrointestinal tract was measured in the range of 670-690 nm under the He-Ne laser (= 632.8 nm) illumination. Surface fluorescence data were collected with the use of a special diagnostic fiber with a total diameter of about 2 mm, which combines some illuminating and receiving fibers. To do the measurements, the diagnostic fiber was brought into a slight contact with the mucosa surface. The quantitative results were calculated with the use of the special fluorescent contrast coefficient (Kf), which takes into account the backscattered light as well. After the fluorescent diagnostic procedure, the low-level laser therapy was applied to all patients. It was found that: first of all, during the laser medical cure the registered fluorescence for the patients with a good dynamics of treatment had an evident tendency to a decrease, and the coefficient Kf for observed ulcers tended to approach the value obtained from normal mucosa (Kf), which indicates the normalization of the porphyrin content in the tissue. Second, the retrospective analysis of the efficacy of laser therapy versus initial values of Kf showed that, for the patients with the initial values Kf = 0.2, the efficiency of the laser treatment was the highest: the relation between the patients with good treatment results and bad ones was 4:1 (more than 80%). For the initial Kf = 0.2-0.3, this relation was 3:1 but for Kf = 0.4-0.5 it was 2:3. The differences in the efficiency of the low-level laser therapy, except the cases of the cancer in the ulcers, may be due to different content of porphyrin in the tissue: at high concentrations of the photosensitizer in the ulcer, the effect of the overdosing could be realized. In this case, another technology of laser illumination is needed. The results are discussed in terms of the free-radical conception of the stimulating effect of laser radiation.

[Phase transition studies of model and biological membranes. I. Use of hydrophobic fluorescent probes for studying phase transitions in liposomes]. / Izuchenie fazovykh perekhodov model'nykh i biologicheskikh membrane. I. Primenenie gidrofobnykh fluorestsentnykh zondov dlia izucheniia fazovykh perekhodov v liposomakh.

Phase transition induced by temperature changes in liposomes prepared from synthetic dimyristoyl-(DML), dipalmytoyl-(DPL) and distearyl-(DSL) lecithins and also from equimolar mixture of DML and DPL and mixtures of DPL and cholesterol were studied with fluorescent probes such as 3-metoxybenzantron (MBA) and dimetylaminocholkone (DMC). The significant changes of fluorescence intensity and maximum position of MBA and DMC were found in the regions of phase transitions at 23, 42, 54 and 31c in liposomes from DML, DPL, DSL and mixture of DML and DPL, respectively. Cholesterol incorporation into liposomes from DPL led to a decrease of transition temperature and cooperativity for these liposomes. At cholesterol concentration of 20 mol. percent or more the transition disappeared completely. It is concluded that MBA and DMC can be used for the investigation of biological membranes phase transitions.

[Temperature-dependent functional changes in sarcoplasmic reticulum membranes]. / Temperaturno-zavisimye funktsional'nye izmeneniia v mebranakh sarkoplazmaticheskogo retikuluma.

Temperature effect on changes in calcium ion transport and activity of Ca-ATPase of sarcoplasmic reticulum vesicles (SRV) was studied. Several temperature intervals of the change of SRV functional activity parameters were found: 8-12 degrees C-beginning of accumulation of SRV Ca2+, 12-19 degrees sharp activation of Ca2+ accumulation, 27-32 degrees-activation of passive yield, 36-43 degrees-reversible change of the activity of the accumulation system and activation of Ca yield, 48 degrees and higher--irreversible denaturation of transport Ca-ATPase. Coincidence of temperature intervals of SRV structural reconstructions, detected by means of fluorescent probes and functional changes points to a close relationship between structural reconstructions and functional responses of biological membranes.

[Effect of valinomycin on structural and functional transitions in sarcoplasmic reticulum membranes]. / Vliianie valinomitsina na strukturnye i funktsional'nye perestroiki membran sarkoplazmaticheskogo retikuluma.

Effect of valinomycin (VAL) on ATPase activity and transport of calcium ions in sarcoplasmic reticulum (SR) vesicles has been studied, as well as the diffusion efflux of calcium ions from liposomes of the total fraction of SR lipids. The studies were carried out in the temperature range of 10 divided by 50 degrees. Changes in the structure of SR membranes were checked with a fluorescent probe of 3-metoxibenzantron. It was shown that at the molar ratios 1:7000-1:70 VAL decreases its ATPase activity and active transport of calcium ions in SR at all temperatures. Besides VAL decreases the rapid efflux of calcium ions from SR and the diffusion efflux of calcium ions from liposomes within the temperature range 10 degrees divided by 35 degrees; it increases the rates of these processes at higher temperatures. Simultaneously the broadening of temperature intervals of structural transitions in the SR membranes takes place.

[Molecular and cellular mechanisms of the low intensity laser radiation effect]. / Molekuliarno-kletochnye mekhanizmy deistviia nizkointensivnogo lazernogo izlucheniia.

The main aspects of the free radical conception of the molecular and cellular mechanisms of the stimulating action of low-intensity radiation in the red region of the spectrum were considered. These are: (1) Primary acceptors of incident radiation are endogenous porphyrins, which may act as photosensitizers giving initiator-radicals for secondary free radical reactions. (2) Target cells for light irradiation during quantum therapy may be blood leukocytes, fibroblasts, keratinocytes, endotheliocytes, etc. (3) The initiation of the secondary free radical reactions due to lipid peroxidation of cell membranes (in particular, of leukocytes) brings about an increase in ion permeability including that for calcium. The increase in intracellular calcium concentration leads to phagocytes priming, i.e., to increased production of reactive oxygen species (ROS) under subsequent stimulation of the cell. (4) Photosensitized generation of ROS in the cytoplasm of some cells induces a free-radical activation of synthesis of proteins, the most significant in the light of the present concept being the de novo synthesis of inducible NO-synthase, superoxide dismutase, and various cytokines. The experimental evidence for the basic statements of the conception of free radical mechanisms for the stimulating action of low-intensity laser and noncoherent radiations is presented. A relation between the primary mechanisms of the stimulating action of light and the secondary effects that determine the sanative effect of quantum therapy in the process of wound healing (bactericidity, cell proliferation, and improved microcirculation) was established. Moreover, it was shown that nitrosyl complexes of heme proteins, such as hemoglobin and cytochrome c, are the primary chromophores of laser radiation. Upon irradiation, they can easily dissociate to produce free nitric oxide. In turn, released nitric oxide may be responsible for blood vessel relaxation and activation of mitochondrial respiration. This phenomenon is just observed during phototherapy by means of low-intensity laser radiation.

[Effect of cholesterol on the physical state and function of lymphocyte membranes]. / Vliianie kholesterina na fizicheskoe sostoianie i funktsionirovanie membran limfotsitov.

Effect of gradual increase of cholesterol content in T-lymphocyte membranes on the structure and physical state of plasmic membrane lipids and activities of the membrane-bound enzymes was investigated. The increase in cholesterol content was shown to result in a two-phase change of luminescence parameters of the fluorescent probes dimethylaminochalcone and pyrene, which indicates heterogeneity of cholesterol in the membranes. With the growth of steroid content in the cell membranes, at first, we observed a sharp decrease in the lipid bilayer fluidity and inhibition of Na+, K+-ATPase activity, which at the molar ratio cholesterol/phospholipids 0.6 in thymocyte membranes, remains at the same level. With higher cholesterol concentrations ATPase activity did not change. The effect of cholesterol on ATPase activity was in a good agreement with the effect of membrane lipids on fluidity. It is suggested that two pools of cholesterol molecules exist in the membranes, differing in their effects of bilayer fluidity and functional activity of the membranes.

[Heterogeneity of phospholipid membranes detected by the fluorescent probe method]. / Geterogennost' fosfolipidnykh membran, vyiavliaemaia metodom fluorestsentnykh zondov.

The measurements of fluorescence polarization index within the emission spectra of the fluorescent probes 4-dimethylaminochalcone (DMCh) and 3-methoxybenzantrone (MBA) have indicated that probes are distributed in liposomes prepared from synthetic dipalmitoyl lecithin (DPL) between binding sites of two types. By the method described in the present paper the spectra measured experimentally have been divided into two spectral components. The shape and position of this componential spectra were independent of the probe:lipid concentrations ratio.

[Effect of valinomycin on the phase state and passive permeability for calcium ions of membranes from artificial phospholipids]. / Vliianie vanilomitsina na fazovoe sostoianie i passivnuiu pronitsaemost' dlia ionov kal'tsiia membran iz iskusstvennykh fosfolipidov.

Effect of valinomycin on passive permeability for calcium ions of liposomes from artificial phospholipids of dimiristoillecithin and dipalmitoillecithin under the conditions of thermotropic structural transitions which were controlled by means of fluorescent probe of 3-metoxibenzantron. It was found that the incorporating of valinomycin into the membrane depends on the density of phospholipid packing. The change of the physical state of phospholipids under the effect of antibiotic was correlated with the change of passive permeability of liposomes for calcium ions.

[Free radical mechanisms of low-intensive laser radiation]. / Svobodnoradikal'nye mekhanizmy stimuliruiushchego deistviia nizkointensivnogo lazernogo izlucheniia.

A hypothesis of free radical mechanisms of stimulating action of low-intensive laser radiation (LILR) used for therapy of a variety of inflammatory diseases is formulated. The main points of the above hypothesis are as follows. Endogenous porphins are a LILR chromofor in the red band (lambda = 632.8 nm). Light absorption induces the production of initiating radicals that are involved in subsequent free radical reactions, in lipid peroxidation in particular. Modified lipid peroxidation in the cell membranes causes an increase in ion permeability, including that for Ca2+. The higher levels of Ca2+ in the leukocytic cytosol result in Ca(2+)-dependent cellular priming, which appeared as the increased cell functional potential and which is seen in subsequent leukocytic stimulation of the greater production of prooxidants and other biologically active products. These products include nitric oxide and a number of cytokines involved in the regulation of microcirculation. The paper presents experimental findings that can be regarded as evidence for some points of the above hypothesis which are used to provide a chain of events underlying the free radical mechanisms of stimulating action of low-intensive laser radiation.