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.

[Stochastic Packaging of Cas Proteins into Exosomes].

CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.

[Metabolic Stress of Red Blood Cells Induces Hemoglobin Glutathionylation].

Metabolic stress caused by a lack of glucose significantly affects the state of red blood cells, where glycolysis is the main pathway for the production of ATP. Hypoglycemia can be both physiological (occurring during fasting and heavy physical exertion) and pathological (accompanying a number of diseases, such as diabetes mellitus). In this study, we have characterized the state of isolated erythrocytes under metabolic stress caused by the absence of glucose. It was established that 24 h of incubation of the erythrocytes in a glucose-free medium to simulate blood plasma led to a two-fold decrease in the ATP level into them. The cell size, as well as intracellular sodium concentration increased. These findings could be the result of a disruption in ion transporter functioning because of a decrease in the ATP level. The calcium level remained unchanged. With a lack of glucose in the medium of isolated erythrocytes, there was no increase in ROS and a significant change in the level of nitric oxide, while the level of the main low-molecular weight thiol of cells, glutathione (GSH) decreased by almost 2 times. It was found that the metabolic stress of isolated red blood cells induced hemoglobin glutathionylation despite the absence of ROS growth. The cause was the lack of ATP, which led to a decrease in the level of GSH because of the inhibition of its synthesis and, probably, due to a decrease in the NADPH level required for glutathione (GSSG) reduction and protein deglutathionylation. Thus, erythrocyte metabolic stress induced hemoglobin glutathionylation, which is not associated with an increase in ROS. This may have an important physiological significance, since glutathionylation of hemoglobin changes its affinity for oxygen.

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.

Search for Intracellular Sensors Involved in the Functioning of Monovalent Cations as Secondary Messengers.

Maintenance of non-equilibrium Na+ and K+ distribution between cytoplasm and extracellular medium suggests existence of sensors responding with conformational transitions to the changes of these monovalent cations' intracellular concentration. Molecular nature of monovalent cation sensors has been established in Na,K-ATPase, G-protein-coupled receptors, and heat shock proteins structural studies. Recently, it was found that changes in Na+ and K+ intracellular concentration are the key factors in the transcription and translation control, respectively. In this review, we summarize results of these studies and discuss physiological and pathophysiological significance of Na+i,K+i-dependent gene expression regulation mechanism.

Heat damage of cytoskeleton in erythrocytes increases membrane roughness and cell rigidity.

The intensity of erythrocyte membrane fluctuations was studied by laser interference microscopy (LIM), which provide information about mechanical properties of the erythrocyte membrane. Atomic force microscopy (AFM) was used to study erythrocyte surface relief; it is related to the cytoskeleton structure of erythrocyte membrane. Intact human erythrocytes and erythrocytes with a destroyed cytoskeleton were used. According to the obtained results, cytoskeleton damage induced by heating up to 50 °Ð¡ results in a reduced intensity of cell membrane fluctuations compared to non-treated cells (20.6 ± 10.2 vs. 30.5 ± 5.5 nm, correspondingly), while the roughness of the membrane increases (4.5 ± 1.5 vs. 3.4 ± 0.5 nm, correspondingly).

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.

Role of Purinergic Receptors of Erythrocytes in the Regulation of Conformation and Oxygen- and NO-Transporting Capacity of Hemoglobin.

Raman spectroscopy was used to investigate changes in hemoglobin conformation and its ability to transfer O2 and NO induced by activation of purinergic receptors of erythrocytes with extracellular ATP. It was found that addition of ATP in a final concentration of 0.1 mM and higher to erythrocyte suspension was followed by changes in hematoporphyrin conformation, while addition of ATP in a concentration of 1 mM and higher increased the proportion of oxyhemoglobin and NO-associated hemoglobin complexes. In case of purinergic receptors activation in blood erythrocytes, significant changes in hemoglobin conformation were observed only at a final ATP concentration of 5 mM, probably due to buffer properties of the blood.

[Insulin-like growth factor 1-induced changes in hemoporphyrin conformation in hemoglobin and NO-transporting function in human erythrocytes].

The evidence on the effect of different insulin-like growth factor concentrations on a state of human erythrocytes is provided. Activation of the insulin-like growth factor receptors led to changes in the conformation of hemoglobin, NO-transporting function in erythrocytes and also in micro-viscosity of the cell membrane. It was shown for the first time that at the binding of the insulin-like growth factor to a cell surface, hemoglobin of erythrocytes alters its conformations and affinity to ligands.

Thermal inactivation of volume-sensitive K⁺,Cl⁻ cotransport and plasma membrane relief changes in human erythrocytes.

Previously, we reported that in mammalian erythrocytes irreversible annealing of spectrin heterodimers at 49-50 °C abolished cell volume-dependent regulation of ion carriers, thus suggesting an implication of a two-dimensional (2D) membrane carcass in volume sensing and/or signal transduction. To further examine this hypothesis, we employed atomic force microscopy. This method revealed folded membrane relief of fixed human erythrocytes with an average wave height of 3-5 nm covered by globular structures with a diameter of 40-50 nm and an average height of 1-2 nm. Erythrocyte swelling caused by reduction of medium osmolality decreased the height of membrane surface waves by 40 % and increased K(+),Cl(-) cotransport by approximately sixfold. Both volume-sensitive changes of membrane relief and activity of K(+),Cl(-) cotransporter were abolished by a 10-min preincubation at 50 °C. Our results strongly suggest that volume-dependent alterations of the human erythrocyte membrane relief are caused by reorganization of the 2D spectrin-actin network contributing to regulation of the activity of volume-sensitive ion transporters.

Changes in the state of hemoglobin in patients with coronary heart disease and patients with circulatory failure.

Morphology of erythrocytes and conformation of hemoglobin-derived hematoporphyrin were studied in patients with coronary heart disease (CHD) and patients with circulatory failure using laser interference microscopy and Raman spectroscopy. Correlation was revealed (r=0.81) between hemoglobin oxygen saturation and oxyhemoglobin fraction in erythrocytes evaluated by Raman spectroscopy. Patients with CHD and patients with circulatory failure showed reduced oxygen-releasing capacity of hemoglobin and hemoglobin content and increased oxygen-binding capacity of hemoglobin, and hemoglobin affinity for oxygen. Significant differences from the control were observed only in patients with circulatory failure. It was found that hemoglobin content, hematocrit, and the shape of erythrocytes during CHD and circulatory failure did not differ from the control, whereas the area of erythrocytes was increased.

[3-Dimensional microscopy as a method for volume measurement in cells undergoing apoptosis].

Different patterns of cell volume perturbations are commonly used for modes of cell death: necrosis (cell swelling) and apoptosis (cell shrinkage). In this study we employed recently developed three dimensional microscopy for the measurement of the volume of attached vascular smooth muscle cells transfected with E1A-adenoviral protein. These cells undergo rapid apoptosis in the absence of growth factors or in the presence of staurosporine. In 30-60 min of serum deprivation the volume of these cells is increased by -40% that corresponds to the time point of maximal activation of caspase 3 and chromatin cleavage. In 10-15 min swollen cells exhibit morphological collapse indicated by formation of apoptotic bodies. In contrast to serum-deprived cells, staurosporine leads to attenuation of cell volume by 30%. In this case, apoptotic bodies are detected in -2.5 hr after maximal shrinkage. Thus, our results show that cell shrinkage can not be considered as universal hallmark of apoptosis. The role of stimulus-specific cell volume perturbation in the triggering of the cell death machinery should be examined further.

[A feedback between membrane fluidity and transcription of the desB gene for the omega3 fatty acid desaturase in the cyanobacterium Synechocystis].

Prokaryotic cells, including cyanobacteria, respond to a decrease in ambient temperature by activation of numerous cold shock genes. Low temperatures cause a decrease in membrane fluidity, which is maintained at some optimal level mainly by fatty acid (FA) desaturases. Here, temperature-dependent expression of the desB gene for the omega3-desaturase in Synechocystis, which synthesized polyunsaturated FAs, and in its mutant, desA-/desD-, which is defective in genes for delta12- and delta6-desaturases and is capable of synthesizing only monounsaturated FAs was studied. Low temperatures caused the increase in the amount of the desB mRNA in the wild-type cells with the maximum observed at 24 degrees C. In the double mutant desA-/desD- cells, the maximum amount of this mRNA was accumulated at 28-30 degrees C. Thus, our studies of the desB gene for the omega3-desaturase demonstrated that temperature-dependent expression of genes, which are responsible for the maintenance of the optimal membrane fluidity, depends on physical state of these membranes and is regulated by a feedback mode.

Oxygen-binding characteristics of erythrocyte in children with type I diabetes mellitus of different duration.

Oxygen-binding properties of erythrocyte hemoglobin were studied in children with type 1 diabetes mellitus by Raman spectroscopy. The content of hemoglobin-oxygen complexes increased significantly only in children with lasting disease (more than 1 year); oxygen-binding capacity of hemoglobin is significantly changed, while its capacity to release oxygen remained unchanged. These changes were paralleled by alteration of hemoglobin affinity for oxygen. The area and content of hemoglobin were studied by laser interference microscopy. Hemoglobin content increased significantly in erythrocytes of patients with a more than 1-year history of type 1 diabetes mellitus. In these children, a significant inverse correlation between oxyhemoglobin fraction, oxygen binding capacity, and cholesterol content was found, this clinical parameter positively correlated with affinity for oxygen measured by Raman spectroscopy.

Laser interference microscopy of amphibian erythrocytes: impact of cell volume and refractive index.

This study examined the action of anisosmotic media on the volume of nucleated erythrocytes isolated from Rana temporaria. Elevation of medium osmolarity from 100 to 345 mOsm resulted in attenuation of mean cell volume by more than 3-fold, estimated by hematocrit measurement. By contrast to this 'classic' erythrocyte volume evaluation technique, we did not observe any significant cell volume modulation by examining the 3D reconstruction of erythrocyte interference images obtained by laser interference microscopy. Comparative analysis of mean cell volume, phase height and cell area appraised by laser interference microscopy showed that the lack of visible alterations of phase image geometry was caused by sharp elevation of the average refractive index of the cytoplasm in shrunken cells. Thus, our results show for the first time that laser interference microscopy in combination with a direct method for cell volume measurement may be employed for estimation of the refractory index of intracellular milieu and for assessment of changes of physical chemical properties of the cytoplasm evoked by diverse stimuli including osmotic stress.

[New possibilities of studying microbial objects by laser interference microscopy].

A novel method, laser interference microscopy, has been developed for studying the morphofunctional state of bacterial cells and the structure of bacterial communities. The following potentialities of the method are shown: rapid determination of the cell structure and subcellular structures (nucleus zone, vacuoles, lamellar structures) and the physiological state of the cell, as well as the study of the structure of bacterial communities (biofilm). The method does not require any additional preparation of cells before the investigation (fixation, staining, treatment with contrasting substances), which reduces the possible appearance of artifacts to a minimum and enables one to use laser interference microscopy for in vivo investigations.

[The use of nanoparticles for the study of conformations submembrane hemoglobin].

The advantages and features of the integrated application of methods of atomic force microscopy, laser interference microscopy and Raman microscopy in the study of erythrocytes was discussed. For the successful application of Raman microscopy in surface enhanced Raman spectroscopy mode the silver colloids was using. The dependence of the enhancement of Raman signals from silver colloids size was demonstrated. The using of developed methods for clinic diagnostic was discussed.

[The study of hypotensive action of dinitrosyl-iron complex with glutathione containing drug oxacom in healthy volunteers].

On the basis of earlier executed studies of hypotensive effect of dinitrosyl iron complexes (DNIC) with glutathione, the drug has been created in industrial conditions named oxacom. Preliminary pharmacological studies of oxacom have not revealed negative qualities. The drug has been now tested in 14 healthy men in whom at single intravenous introduction it caused typical response - a decrease of diastolic as well as systolic arterial pressure on 24-27 mmHg through 3-4 min with subsequent very slow restoration in 8-10 hours. The heart rate after initial rise was quickly normalized. Echocardiography revealed unaltered cardiac output in spite of reduced cardiac filling by 28%. The multilateral analysis of clinical and biochemical data has revealed an absence of essential alterations which could lead to pathological consequences. The drug is recommended for carrying out of the second phase of clinical trial. The comparative study of the efficiency of hypotensive action of oxacom, S-nitrosoglutathione (GS-NO) and sodium nitrite (NO2) in rats has shown that the duration of effect was the greatest at oxacom action.

[Studies of the blood antioxidant system and oxygen-transporting properties of human erythrocytes during 105-day isolation].

Effects of strict 105-d isolation on blood antioxidant status, erythrocyte membrane processes and oxygen-binding properties of hemoglobin were studied in 6 male volunteers (25 to 40 y.o.) in ground-based simulation of a mission to Mars (experiment Mars-105). The parameters were measured using venous blood samples collected during BDC, on days 35, 70 and 105 of the experiment and on days 7 and 14-15 after its completion. Methods of biochemistry (determination of enzyme activity and thin-layer chromatography) and biophysical (laser interference microscopy, Raman spectroscopy) showed changes in relative content of lipid and phospholipid fractions suggesting growth of membrane microviscosity and increase in TBA-AP (active products of lipids peroxidation interacting with thiobarbituric acid). A significant increase in glucose-6-phosphate dehydrogenase and superoxide dismutase activities against reduction of catalase activity points to both reparative processes in erythrocytes and disbalance between the number of evolving active forms of oxygen and antioxidant protection mechanisms in cells. Hemoglobin sensitivity of oxygen and blood level of oxyhemoglobin were found to increase, too. It is presumed that adaptation of organism to stresses experienced during and after the experiment may destroy balance of the antioxidant protection systems which is conducive to oxidation of membrane phospholipids, alteration of their content, increase of membrane microviscosity and eventual failure of the gas-exchange function of erythrocytes.

Na+ i/K+ i imbalance contributes to gene expression in endothelial cells exposed to elevated NaCl.

High-salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodelling, cardiac hypertrophy and stroke incidence. Alterations in NO production, inflammation and endothelial cell stiffening are considered now as plausible mediators of cardiovascular dysfunction. We studied early responses of endothelial cells (HUVEC) caused by a moderate increase in extracellular sodium concentration. Exposure of HUVEC to elevated sodium within the physiological range up to 24 h is accompanied by changes in monovalent cations fluxes and Na,K-ATPase activation, and, in turn, results in a significant decrease in the content of PTGS2, IL6 and IL1LR1 mRNAs. The expression of NOS3 and FOS genes, as well as the abundance of cytosolic and nuclear NFAT5 protein, remained unchanged. We assessed the mechanical properties of endothelial cells by estimating Young's modulus and equivalent elastic constant using atomic force and interference microscopy, respectively. These parameters were unaffected by elevated-salt exposure for 24 h. The data obtained suggest that even small and short-term elevations of extracellular sodium concentration affect the expression of genes involved in the control of endothelial function through the Na+ i/K+ i-dependent mechanism(s).