Spectral and conformational characteristics of phycocyanin associated with changes of medium pH.

C-phycocyanin (C-PC) is the main component of water-soluble light-harvesting complexes (phycobilisomes, PBS) of cyanobacteria. PBS are involved in the absorption of quantum energy and the transfer of electronic excitation energy to the photosystems. A specific environment of C-PC chromophoric groups is provided by the protein matrix structure including protein-protein contacts between different subunits. Registration of C-PC spectral characteristics and the fluorescence anisotropy decay have revealed a significant pH influence on the chromophore microenvironment: at pH 5.0, a chromophore is more significantly interacts with the solvent, whereas at pH 9.0 the chromophore microenvironment becomes more viscous. Conformations of chromophores and the C-PC protein matrix have been studied by Raman and infrared spectroscopy. A decrease in the medium pH results in changes in the secondary structure either the C-PC apoproteins and chromophores, the last one adopts a more folded conformation.

Changes in the Frequency of Rhythmic Excitation of Retzius Cells during Thermal Stimulation of Leech Skin.

Thermal stimulation of various parts of the skin in Hirudo medicinalis increases the frequency of spontaneous rhythmic excitation of Retzius neurons in leech ganglia. It was shown that the frequency of spontaneous rhythmic excitation of Retzius cells in the segmental ganglion increases only in response to thermal stimulation and returns to initial values upon cooling. This effect was also detected in neurons that are not directly connected by nerve fibers with the particular skin area. Changes in the frequency of spontaneous rhythmic excitation of Retzius cells in the segmental ganglion were observed during thermal stimulation of not only leech body, but also of the head and caudal suckers. These changes in spontaneous rhythmic excitation of Retzius cells in the segmental ganglion during thermal stimulation were observed in Hirudo medicinalis, but not in Macrobdella decora.

Visualization and Cytotoxicity of Fluorescence-Labeled Dimeric Magnetite-Gold Nanoparticles Conjugated with Prostate-Specific Membrane Antigen in Mouse Macrophages.

We demonstrated the possibility of penetration of magnetite-gold nanoparticles conjugated with prostate-specific membrane antigen into mouse macrophages. It was found that after 3-h incubation with nanoparticles in a concentration of 15 mg/liter at 37oC, they were seen in only 13% macrophages. In about 90% cells, the nanoparticles were detected within the cytoplasm. Under these conditions, membrane damage was revealed in 25% cells. These results should be taken into account in further development and application of nanomaterials for diagnostic and therapeutic purposes in oncology.

[Effect of the incubation medium pH on damage to macrophages plasma membranes].

In this paper we showed the pH-dependent change in the sensitivity of the membranes of murine peritoneal macrophages to UV-radiation. This relationship is discussed in terms of lipid bilayer membrane stability modification to the action of ROS and lipid peroxidation process (LPP) at different pH. Iron-ascorbate reinforced LPP also led to pH-dependent membranes damage. The increase of the cells incubation medium temperature up to 37 degrees C, which also stimulated LPP, did not change the picture of the pH-dependent damage. Decrease of the incubation medium pH did not reduce H2O2-induced cell damage. Increase of the pH intensified the cells damage.

[Effect of hydroxylated fullerene C60(OH)25 on macrophage plasma membrane integrity].

Our study has shown that the damaging effect of hydroxylated fullerene C60(OH)25 on mouse peritoneal macrophage plasma membranes increased when we enlarged the concentration of fullerene in the incubation media (from 0.005 to 0.5 mg/ml), the incubation temperature (from 22 degrees C to 37 degrees C) and the time of incubation (from 30 to 90 min). In conditions of the H2O2-induced membrane damage, fullerene was observed to intensify the H2O2-induced damaging effect at a concentration of 0.05 mg/ml and reduce it at a concentration of 0.5 mg/ml. In conditions of the UV-induced membrane damage, it was discovered that the damaging effect of UV increased when C60(OH)25 nanoparticles were added to the incubation media before irradiation and decreased when they were added after irradiation. Eventual participation of ROS in damaging effects of C60(OH)25 was discussed.

[Damaging effect of "Poviargol" on mice peritoneal macrophage plasma membrane].

The damaging effect of "Poviargol", a substance containing silver nanoparts, was studied. It was shown that the damaging effect of "Poviargol" took place from the concentration of 2 mkg/ml and got its maximum at 10-12 microg/ml. Decrease of the incubation temperature from 30 to 4 degreesC led to amplification of the membrane-acting effect of "Poviargol"; however, inverse relation was observed in the range from 37 to 30 degreesC. The damaging effect of "Poviargol" increased when pH of the incubating medium was raised to 8.4 and also when the concentration of calcium ions in the incubation medium was raised to 8 mmol/l. The damaging effect decreased when pH of the incubation medium was reduced to 6.3, as well as in the presence of radioprotector serotonin. Our study allows us to suppose that reactive oxygen species and lipid peroxidation make a substantial contribution to the damaging effect of "Poviargol" on the macrophage plasma membrane.

[Alteration of UV-sensitivity of mice peritoneal macrophage plasma membranes by calcium ions].

Effect of Ca2+ ions on UV-induced mice peritoneal macrophage plasma membrane damage has been studied. Drop of the extracellular Ca2+ concentration has been found to result in a reduced expression of this damage. On the contrary, a raised intra- and extra-cellular Ca2+ level is associated with a higher number of cells with damaged plasma membranes. These findings make it possible to suggest that this change in the plasma membrane photosensitivity might be a result of alterations in the membrane lipid matrix electrical stability owing to UV-induced lipid photo-peroxidation. This study has also shown that free radical peroxidation of membrane lipids plays a significant part in UV-induced cell damage.

[Application of laser interference microscopy (LIM) for investigating the features of UV(B)-irradiated mouse peritoneal macrophages].

The plasma membrane dose-dependent damage of UV(B)-irradiated mouse peritoneal macrophages was investigated using laser interference microscopy (LIM). LIM is a method which allows one to estimate morphological and functional parameters of a cell without dyeing or introduction of other substances which can affect the cell condition. This makes it possible to reduce and accelerate the procedure of counting the damaged cells as compared with the methods using different dyes. The value of optical path difference (OPD)--a variable proportional to the object thickness and the difference in the refractive indices of the object and the surrounding medium was used for estimation of the cell damage. Also compared was usability of LIM and microfluorimetry assay in investigations of the UV(B)-irradiated macrophage plasma membrane.

[The influence of hydrogene ion concentrations on peritoneal macrophage plasma membrane's photosensitivity].

It was demonstrated on macrophages treated with UV irradiation dose 9 J/sm2 (lamda max =306 nm) that intra- or extracellular pH reducing lead to decrease the number of cells with damaged membranes in macrophage population. An intra- or extracellular pH elevation leads to increase of UV-irradiation membrane-acting effect. It was also shown that pH-dependence of UV-irradiation damage effect has been lost after preliminary osmotic swelling of cells. The cells survived after UV-irradiation in doses 8 and 10 J/sm2 (lamda max =297 nm) have an intracellular pH lower than non-irradiated cells.

[Damaging effect of digitonin on macrophage plasma membranes exposed to UV-irradiation].

It was shown that macrophage irradiation in 4.6 J/cm2 (lambda(max) = 306 nm) dose leads to small quantity of damaged cells in cell population, which doesn't change substantially during 60 min of incubation in darkness. So as detergent digitonin treatment (without irradiation) in 3 mkg/ml concentration doesn't lead to substantial cell damage. Also the result of combined influence of UV-irradiation and digitonin added after irradiation, 15 min before the damaged cells counting, has been got. It was shown that macrophage incubation for 15 minutes leads to cell damaging twice as much sum of UV (4.6 J/cm2) and digitonin (3 mkg/ml) damaging. However the level of cell damaging obtained 30 minutes later after finishing of irradiation doesn't exceed the sum of separate effects of this factors. Further increase of postradiation time leads to synergic effect again.

[Damage to macrophage membranes by exposure to middle-wave ultraviolet radiation]. / Povrezhdenie membran makrofagov pri deistvii srednevolnovogo ul'trafioletovogo izlucheniia..

An influence of middle-wave ultraviolet radiation (lambda max = 306 nm) on plasma membranes of mice peritoneal macrophages was studied by microfluorimetry analysis. It was found that a percentage of cells with damaged plasma membranes in the irradiated macrophage population reliably increased with the UVB dose starting with 6 J/cm2. Irradiation of cells with 4.2 J/cm2 UVR dose which does not cause evident damage to plasma membranes led to the latent damage which was detected by treatment with detergent digitonin (4.5 micrograms/ml).

[Modification of damaging effect of ultraviolet radiation on peritoneal macrophage membranes in mice]. / Modifikatsiia povrezhdaiushchego deistviia ul'trafioletovogo izlucheniia na membrany peritoneal'nykh makrofagov myshei.

Different factors modificating damaging effect of middle-wave ultraviolet radiation (lambda max = 306 nm) on mice peritoneal macrophage plasma membranes were studied. It was shown that damaging effect of ultraviolet declined when the cells were simultaneously treated by red light (lambda max = 713 nm). This protective effect increased when a red component of light became greater and achieved almost 100% when it consisted 28.6%. It was also found that the decrease in intra- and extracellular pH led to the decrease in damaging effect of UVR. The increase of pH bring to an elevation of UVR destructive effect.

[Influence of Ca2+ ions on UV-induced damage to mice peritoneal macrophage plasma membranes]. / Vliianie ionov na UF-indutsirovannoe povrezhdenie plazmaticheskikh membran peritoneal'nykh makrofagov myshei.

It was shown that UV-irradiation caused damage to mice peritoneal macrophage plasma membranes. A decrease in extracellular Ca2+ leads to a decrease of the damaging effect. An increase in extracellular Ca2+ or adding of calcium ionophore A23187 to the medium is accompanied by an increase in a number of damaged cells. These data allow us to suppose that modification of the damaging effect of UV-irradiation by Ca2+ ions can be bound with changing of electric stability of membrane lipid matrix.

[Influence of intracellular pH on character of mice peritoneal macrophage response to red light irradiation]. / Vliianie vnutrikletochnogo pH na kharakter otveta peritoneal'nykh makrofagov pri deistvii krasnogo sveta.

It was detected that exposure of macrophages to red light (600-740 nm) led to the changes in their intracellular pH and hydrolytic activity. The character of these changes depends on the initial level of pH in the cells. The maximum effect of irradiation is detected if the initial pH level is low. It is possible that Na/H-exchanger takes part in normalizing effect of red light on intracellular pH level.

[Alteration of intracellular pH of macrophages after UV-irradiation]. / Izmenenie vnutrikletochnogo pH makrofagov posle UF-oblucheniia.

It was shown that in vitro exposuse of mice peritoneal middle-wave ultraviolet radiation (lambdamax = 306 nm) in doses which don't damage to cause plasma membrane caused dose-dependent decreasing of their intracellular pH. After exposure of cells to 0.5 J/cm2 it was detected an acidification of intracellular contents followed by an increase of intracellular pH up to control level (after 40 min of incubation) and then above it (on 45 min of incubation). An increase of irradiation dose was accompanied by more evident reduction of intracellular pH and lack of its restoration on 45 min of postradiational incubation under irradiation with a dose of 3 J/cm2.

Study of conductance changes of bilayer lipid membrane induced by electric field.

Changes in the bilayer lipid membrane (BLM) conductance induced by electric field were studied. BLMs were formed from diphytanoylphosphocholine (DPhPC) solution in squalene. Certain time after a constant voltage (200-500 mV) was applied to the BLM in the voltage-clamp mode, the BLM conductance started to grow up to approximately 10 nS until the BLM ruptured. The conductance often changed abruptly (with the front duration of less than 33 micros) and then stabilized for a relatively long time (up to 10; 300 ms on average) thus resembling the ion channel activity. The mean amplitude of conductance steps was 650 pS. However, in some cases a slow conductance drift was recorded. When N-methyl-D-glucamine/glutamate ions were used instead of KCl, the conductance changes became 5 times smaller. We suggest that formation in the BLM of single pores approximately 1 nm in diameter should result in the observed changes in BLM conductance. The BLM conductance growth was due to consecutive opening of several such pores. When the electric field amplitude was abruptly decreased (down to 50-100 mV), the conductance dropped rapidly to the background value. When we increased the voltage again, the BLM conductance right after the increase depended on the time BLM spent under "weak" electric field. If this time exceeded 500 ms, the conductance was at the background level, but when the time was diminished, the conductance reached the value recorded before the voltage decrease. These data imply that the closure of the pores should lead to the formation in BLM of small defects (prepores) that can be easily transformed into pores when the voltage is increased. The lifetimes of such prepores did not exceed 500 ms.