ABSTRACT
The qualitative composition and zeta potential of magnetite nanoparticles (size 4.2±1.2 nm) obtained by co-precipitation method were determined by X-ray and diffraction dynamic light scattering. The zeta potential of Fe3O4 particles was -15.1±4.5 mV. The possibility of interaction of magnetite nanoparticles with human blood plasma proteins and hemoglobin as well as with erythrocyte membranes was demonstrated by spectrophotometry, electrophoresis, and fluorescence methods. No changes in the sizes of hemoglobin molecules and plasma proteins after their modification by Fe3O4 particles were detected. The possibility of modifying the structural state of erythrocyte membranes in the presence of magnetite nanoparticles was demonstrated by means of fluorescent probe 1-anilinonaphthalene-8-sulfonate.
Subject(s)
Hemoglobins , Magnetite Nanoparticles , Humans , Magnetite Nanoparticles/chemistry , Hemoglobins/chemistry , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/chemistry , Erythrocyte Membrane/metabolism , Particle Size , Blood Proteins/chemistry , Anilino Naphthalenesulfonates/chemistry , X-Ray Diffraction , Ferrosoferric Oxide/chemistry , Fluorescent Dyes/chemistryABSTRACT
The article reviews the results of the studies of marker parameters (indicators) of various pathways and mechanisms of apoptosis of lymphocytes in donor peripheral blood induced by UV light (240-390 nm) in doses of 151, 1510, and 3020 J/m2. The article analyses the processes of DNA fragmentation, distortion of the structural asymmetry of the cell membranes, changes in the degree of DNA damage (single-strand breaks), transcriptional factor Ñ53, cytochrome Ñ, Fas receptors (CD95), caspase-3, caspase-8, and caspase-9, reactive oxygen species, and calcium ions in UV modified cells. The study determined that programmed cell death of lymphocytes after UV irradiation with 1510 J/m2 involves the Ñ53-dependent pathway of the nuclear mechanism, as well as receptor-mediated caspase mechanism, mitochondrial mechanism, and the mechanism associated with the defects in calcium homeostasis. Cell death is mediated by reactive oxygen and calcium ions. The article suggests a scheme of possible intracellular events resulting in the apoptotic death of lymphocytes after UV irradiation.
ABSTRACT
In today's world, there is a wide array of materials engineered at the nano- and microscale, with numerous applications attributed to these innovations. This review aims to provide a concise overview of how nano- and micromaterials are utilized for enzyme immobilization. Enzymes act as eco-friendly biocatalysts extensively used in various industries and medicine. However, their widespread adoption faces challenges due to factors such as enzyme instability under different conditions, resulting in reduced effectiveness, high costs, and limited reusability. To address these issues, researchers have explored immobilization techniques using nano- and microscale materials as a potential solution. Such techniques offer the promise of enhancing enzyme stability against varying temperatures, solvents, pH levels, pollutants, and impurities. Consequently, enzyme immobilization remains a subject of great interest within both the scientific community and the industrial sector. As of now, the primary goal of enzyme immobilization is not solely limited to enabling reusability and stability. It has been demonstrated as a powerful tool to enhance various enzyme properties and improve biocatalyst performance and characteristics. The integration of nano- and microscale materials into biomedical devices is seamless, given the similarity in size to most biological systems. Common materials employed in developing these nanotechnology products include synthetic polymers, carbon-based nanomaterials, magnetic micro- and nanoparticles, metal and metal oxide nanoparticles, metal-organic frameworks, nano-sized mesoporous hydrogen-bonded organic frameworks, protein-based nano-delivery systems, lipid-based nano- and micromaterials, and polysaccharide-based nanoparticles.
ABSTRACT
We studied the effect of amphotericin B (2.5×10-5 and 5.4×10-5 M) on osmotic resistance and surface cytoarchitectonics of donor blood erythrocytes. Antibiotic at a concentration of 2.5×10-5 M induced most pronounced changes in the studied parameters, which can be related to the specifics of the spatial organization of the cholesterol-amphotericin B complexes at different stoichiometric ratios of the components and their ability to pore formation in the membranes. Cholesterol binding to the polyene antibiotic and the appearance of perforations in the plasma membrane lead to accumulation of reversibly and irreversibly deformed cells and their hemolysis. The appearance of a large number of irreversibly deformed erythrocytes indicates an impaired ability to elastic deformation in the microcirculatory stream, which can lead to disruption of their functions in vivo and intravascular hemolysis.
Subject(s)
Amphotericin B , Anti-Bacterial Agents , Humans , Amphotericin B/pharmacology , Amphotericin B/chemistry , Anti-Bacterial Agents/pharmacology , Microcirculation , Polyenes/analysis , Polyenes/pharmacology , Erythrocytes , Cholesterol , HemolysisABSTRACT
The effect of UV-light (240-390 nm) in doses of 151 and 755 J/m2 on the expression of membrane markers CD5, CD19, CD20 in human peripheral blood B cells was studied by flow cytometry. In 24 h after exposure to UV light, we observed activation of processes accompanied by structural rearrangements of B-cell membranes leading to changes in the expression of receptor molecules: the content of of CD19 and CD20 increased due to activation of the synthesis of these proteins, while the content of CD5 decreased. The percentage of CD5+ cells decreased over 24 h after UV-irradiation of lymphocytes, while addition of autologous plasma to the incubation medium produced a photoprotective effect on CD5+ cells.
Subject(s)
Antigens, CD , B-Lymphocytes , Blood Transfusion, Autologous , Antigens, CD/metabolism , Antigens, CD/radiation effects , Antigens, CD19/metabolism , Antigens, CD19/radiation effects , Antigens, CD20/metabolism , Antigens, CD20/radiation effects , B-Lymphocytes/metabolism , B-Lymphocytes/radiation effects , Biomarkers/metabolism , CD5 Antigens/metabolism , CD5 Antigens/radiation effects , Cell Membrane/metabolism , Cell Membrane/radiation effects , Humans , Immunity, Cellular/radiation effects , Immunotherapy/methods , Ultraviolet Rays/adverse effectsABSTRACT
Exposure of human lymphocytes to silver nanoparticles (2-12 nm) reduced viability of cells and RNA, reduced activities of lactate dehydrogenase, glutathione reductase, and cytosolic calcium, increased ROS content in cells, affected surface architectonics of cells and changed hydrophobicity and charge of their plasma membranes. Silver nanoparticles triggered the process of cellular death by the mechanism of ETosis that is accompanied by chromatin release into the extracellular environment and formation of extracellular traps.
Subject(s)
Lymphocytes/metabolism , Metal Nanoparticles/chemistry , Silver/chemistry , Adolescent , Adult , Calcium/metabolism , Extracellular Traps/metabolism , Glutathione Reductase/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , Male , Microscopy, Fluorescence , Reactive Oxygen Species/metabolism , Young AdultABSTRACT
The effects of ROS on functional properties (cytotoxic activity, antibody-producing activity, TNFα synthesis, and free cytosol calcium level), membrane structure (by expression of some surface markers), and apoptosis of lymphocytic cells were estimated in the peripheral blood of healthy volunteers. 1O2, [Formula: see text], OHâ¢, and H2O2 mostly suppressed cytotoxic activity of lymphocytes against Ehrlich ascites carcinoma cells and inhibited IgG synthesis and expression of receptors and surface markers (Fc receptors, CD3, CD19, and CD56). The exposure of lymphocytes to H2O2 (10-6 M), 1O2, and OH⢠was followed by an increase in the level of a secondary messenger, intracellular calcium, in comparison with non-exposed cells. The presence of exogenous calcium in the medium for lymphocyte suspending induced an increase in the number of cells at early and late stages of apoptosis 6 h after exposure to H2O2 and 1O2 in comparison with lymphocytes incubated in Ca2+-free medium.
Subject(s)
Lymphocytes/drug effects , Lymphocytes/metabolism , Reactive Oxygen Species/metabolism , Adolescent , Adult , Apoptosis/drug effects , Calcium/metabolism , Cells, Cultured , Healthy Volunteers , Humans , Hydrogen Peroxide/pharmacology , Hydroxyl Radical/pharmacology , Male , Middle Aged , Singlet Oxygen/pharmacology , Young AdultABSTRACT
Scanning electron microscopy study showed that exposure to CO for 60, 75, and 90 min induced heterogeneous changes in erythrocyte population. Increasing the duration of exposure of blood erythrocytes to CO was followed by the appearance of cells with morphological changes. The formation of discocytes with processes (≥1) was followed by the appearance of "deflated ball"-shaped erythrocytes. Moreover, CO modulated activity of glucose-6-phosphate dehydrogenase in human erythrocytes and disturbed their energy metabolism (suppressed lactate dehydrogenase activity in forward reaction and increased it in reverse reaction). A significant decrease in the coefficient of energy metabolism of erythrocytes (from 36±14 to 5.0±2.5 arb. units) reflected metabolic maladaptation induced by the exposure to CO.
Subject(s)
Carbon Monoxide/pharmacology , Energy Metabolism , Erythrocytes/metabolism , Adolescent , Adult , Catalysis , Erythrocytes/cytology , Female , Glucosephosphate Dehydrogenase/metabolism , Healthy Volunteers , Humans , L-Lactate Dehydrogenase/metabolism , Male , Microscopy, Electron, Scanning , Middle Aged , Surface Properties , Time Factors , Young AdultABSTRACT
The enzymatic hydrolysis of poly- and oligosaccharides from plants seems like an advantageous approach for sugars production. Two inulinases producing fructose from plant oligosaccharides were isolated from yeast Kluyveromyces marxianus and plant Helianthus tuberosus. Both enzymes were immobilized on polymeric carriers by using the static adsorption approach. We could save 80.4% of the initial catalytic activity of plant inulinase immobilized on KU-2 cation-exchange resin and 75.5% of yeast enzyme activity adsorbed on AV-17-2P anion-exchange resin. After immobilization, the Km values increased 1.5 and 6 times for enzymes from K. marxianus and H. tuberosus, respectively. The optimal temperatures for catalysis of both enzymes were increased from 48-50⯰C up to 70⯰C. The activities of both immobilized enzymes remained unchanged after the 10â¯cycles of 20-min hydrolysis reaction at 70⯰C model batch reactor. Sorbents, native and immobilized enzymes did not exhibit any mutagenic or cytotoxic activity.
Subject(s)
Fructose/chemistry , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Helianthus/enzymology , Kluyveromyces/enzymology , Plant Extracts/chemistry , Adsorption , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Enzymes, Immobilized/toxicity , Glycoside Hydrolases/toxicity , Humans , Hydrolysis , Inulin/chemistry , MCF-7 Cells , Resins, Synthetic/chemistryABSTRACT
We studied the effect of carbon monoxide (60-, 75-, and 90-min exposure) on the expression of antiapoptotic proteins (survivin and Bcl-2) in human blood lymphocytes in the presence of recombinant IL-2 in an apoptosis-inducing dose (0.1 ng/ml). Incubation of cells in atmosphere with carbon monoxide in the presence of recombinant IL-2 was accompanied by accumulation of Bcl-2 protein with simultaneous decrease of survivin content. It was concluded that carbon monoxide plays a role in the dysregulation of apoptosis of human blood lymphocytes Bcl-2 (i.e. CO inhibits the proapoptotic effect of recombinant IL-2).
Subject(s)
Apoptosis/drug effects , Carbon Monoxide/pharmacology , Inhibitor of Apoptosis Proteins/genetics , Interleukin-2/pharmacology , Lymphocytes/drug effects , Proto-Oncogene Proteins c-bcl-2/genetics , Apoptosis/genetics , Gene Expression Regulation , Humans , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Inhibitor of Apoptosis Proteins/metabolism , Interleukin-2/antagonists & inhibitors , Lymphocytes/cytology , Lymphocytes/metabolism , Primary Cell Culture , Proto-Oncogene Proteins c-bcl-2/agonists , Proto-Oncogene Proteins c-bcl-2/metabolism , Recombinant Proteins/pharmacology , Signal Transduction , SurvivinABSTRACT
We investigated the mechanism of UV-radiation influence on trypsin in free and immobilized (on chitosan) states. The catalytic activity of free enzyme under the action of UV-light is subjected to changes to a greater extent than that of the immobilized one. We assume that the photoprotection effect of chitosan is caused for the following reasons: firstly, through interactaction with trypsin molecules chitosan forms a more photoresistant complex as compared to the native protein; secondly, chitosan probably binds the active photopro- ducts of a free radical nature, thus preventing oxidation (destruction) of several amino acids of the enzyme under its UV-radiation.
Subject(s)
Chitosan/chemistry , Enzymes, Immobilized/chemistry , Trypsin/chemistry , Ultraviolet Rays/adverse effects , Amino Acids/chemistry , Amino Acids/radiation effects , Enzymes, Immobilized/radiation effects , Free Radicals/chemistry , Hydrogen-Ion Concentration , Oxidation-Reduction/radiation effects , Radiation Protection , Trypsin/radiation effectsABSTRACT
Human oxyhemoglobin exhibits high resistance to nitroglycerin during incubation of the protein with this compound for 0.3-3 h. Prolonged exposure (24 h) leads to activation of methemoglobin production. In the presence of nitroglycerin hemoglobin molecules undergo rapid oxidation during deoxygenation with formation of methemoglobin as the terminal product of human oxyhemoglobin interaction with nitroglycerin. The scheme of interaction processes of oxyhemoglobin with nitroglycerin in different conditions of oxygen regime is proposed. Partially deliganded hemoglobin plays the leading role in the initiation of hemoglobin oxidation processes.
Subject(s)
Methemoglobin/metabolism , Nitroglycerin/pharmacology , Oxygen/metabolism , Oxyhemoglobins/metabolism , Humans , Methemoglobin/chemistry , Nitroglycerin/chemistry , Oxidation-Reduction , Oxygen/chemistry , Oxyhemoglobins/chemistry , Protein Binding/drug effectsABSTRACT
The influence of UV-light (240-390 nm) at dozes of 151 and 755 J/m2 on the content of membrane markers of lymphocytes using the method of flow cytometry was investigated. It was demonstrated that during incubation of UV-irradiated lymphocytes the change of their populational and sub-populational composition occurs. Expression of complexes of CD3, CD 19,.CD8, CD 16, CD25 and CD95 increased. This increase was caused mainly by de novo synthesis. UV-light had immunostimulating effect on CD8+ T-lymphocyte population. Together with the increase of cytotoxic cells and NK-cells, activation of lymphocytes (increased amount of CD25+ and CD95+ cells) took place. Amount of cells undergone apoptosis or necrosis increased proportionally to the dosage. These changes were more expressed during incubation of lymphocytes in nutrition medium without autological blood serum, e.g. under deficiency of growth factors and antioxidants.
Subject(s)
Antigens, CD/radiation effects , Apoptosis/radiation effects , Biomarkers/blood , Lymphocytes/radiation effects , Antigens, CD/blood , Blood Donors , Flow Cytometry , Humans , Killer Cells, Natural/metabolism , Killer Cells, Natural/radiation effects , Lymphocytes/metabolism , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/radiation effects , Ultraviolet RaysABSTRACT
Using computer modeling, virtual screening of high-affinity ligands for immobilization of inulinase - an enzyme that cleaves inulin and fructose-containing polymers to fructose - has been performed. The inulinase molecule from Aspergillus ficuum (pdb: 3SC7) taken from the database of protein structures was used as a protein model and the target for flexible docking. The set of ligands studied included simple sugars (activators, inhibitors, products of enzymatic catalysis), as well as high-molecular weight compounds (polycation and polyanion exchange resins, glycoproteins, phenylalanine-proline peptide, polylactate, and caffeine). Based on the comparative analysis of the values of the total energy and the localization of ligand binding sites, we made several assumptions concerning the mechanisms of interaction of the suggested matrices for the immobilization of enzyme molecules and the structural features of such complexes. It was also assumed that the candidates for immobilization agents meeting the industrial requirements may be glycoproteins, for which we propose an additional incorporation of cysteine residues into their structure, aimed to create disulfide «anchors¼ to the surface.
Subject(s)
Aspergillus/enzymology , Computer Simulation , Fungal Proteins/chemistry , Glycoside Hydrolases/chemistry , Molecular Docking Simulation , Binding SitesABSTRACT
The spectral and oxygen-binding characteristics of human intracellular hemoglobin in the presence of nitroglycerin at concentrations of 5 ng/mL and 5 µg/mL have been studied. Short incubation (20 min) of erythrocytes with the drug led increasing hemoglobin affinity to oxygen and weakening of cooperative interactions in hemoprotein molecules. As a result, the amount of O(2) supplied to tissues in the process of gas exchange decreased by 23.96% (5 ng/mL) and 26.68% (5 µg/ml), p < 0.05. Incubation of cells for 24 h resulted in oxidation of the heme iron atom, accumulation of methemoglobin, and partial hemolysis. Nitroglycerin reduces the intensity of oxidative processes. However, no dependence of the degree of changes in the physical and chemical properties of hemoglobin on the concentration of nitroglycerin was found.
Subject(s)
Erythrocytes/metabolism , Nitroglycerin/pharmacology , Oxygen/metabolism , Oxyhemoglobins/metabolism , Dose-Response Relationship, Drug , Erythrocytes/chemistry , Humans , Oxygen/chemistry , Oxyhemoglobins/chemistry , Spectrum AnalysisABSTRACT
UV-induced changes in the catalytic activity and radiuses of inulinases molecules from various producers (plants, fungy, yeast) are studied. It is established that specific enzymes activity and the sizes of inulinases molecules from Helianthus tuberosus and Kluyveromyces marxianus under the influence of UV-light in the ranges of doses 4530-6040 and 755-6040 J/m2, respectively, are subjected to changes more than structural and functional characteristics of inulinase fromAspergillus niger. It is probably connected with lower contents in it of aromatic amino acids such as tyrosine and phenylalanine. The most expressed loss of functional properties of inulinase from Helianthus tuberosus can be caused by the'existence of significantly more numbers of cysteine in plant fructan-exohydrolases in relation to microbic enzymes. A scheme for the stages of response of inulinases of various origins on the influence of UV-light in a certain range of radiation doses is offered.
Subject(s)
Aspergillus niger/radiation effects , Glycoside Hydrolases/metabolism , Helianthus/radiation effects , Kluyveromyces/radiation effects , Radiation Monitoring/methods , Ultraviolet Rays , Aspergillus niger/enzymology , Dose-Response Relationship, Radiation , Glycoside Hydrolases/chemistry , Helianthus/enzymology , Kluyveromyces/enzymology , Radiation ToleranceABSTRACT
The computer model for a dimer of inulinase from Aspergillus ficuum is designed. The permolecular organization of inulinase from Aspergillus niger is experimentally investigated. The question about the role of various inulinase forms in manifestation of their functional activity is discussed. It is shown, that in the process of inulinase dimerization when contacts between monomeric forms of the enzyme are formed, a key role belongs to the nonpolar amino acid residues.
Subject(s)
Amino Acids/chemistry , Aspergillus/chemistry , Fungal Proteins/chemistry , Glycoside Hydrolases/chemistry , Inulin/chemistry , Aspergillus/enzymology , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Static Electricity , ThermodynamicsABSTRACT
The decomposition pathways of hemoglobin UV absorption spectrum into the absorption spectra of the protein and non-protein components are proposed and substantiated by means of an additive model. We have established that the heme component has an absorption band with a maximum at λ(max) = 269.2 nm (ε = 97163) and the apoprotein component has an absorption band with a maximum at λ(max) = 278.4 nm (ε = 48669) for the wavelength range from 240.0 to 320.0 nm. An integral relative proportion of absorption for the heme fraction (78.8%) and apoprotein (21.2%) in the investigating wavelength range is defined.
Subject(s)
Amino Acids/chemistry , Apoproteins/chemistry , Hemoglobins/chemistry , Models, Theoretical , Spectrum Analysis , Ultraviolet RaysABSTRACT
The flow-cytometric analysis of condition of human peripheral blood lymphocytes in dynamics of development of the apoptosis induced by exposure to UV-light (240-390 nanometers) at a dose of 1510 J/m2 was carried out. Superficial architectonics and changes of the level of a membrane potential and functional activity of succinatedehydrogenase of mitochondrions are studied. The contribution of extracellular calcium to the processes of lymphocyte cellular death is revealed. The ideas about dynamics of the intracellular events leading to the death of photomodified lymphocytes 1-6 h after their UV-radiation exposure are specified and added on the basis of the analysis of our new and already published results.
Subject(s)
Apoptosis/radiation effects , DNA Fragmentation/radiation effects , Lymphocytes/radiation effects , Ultraviolet Rays , Calcium/metabolism , Cell Culture Techniques , Cells, Cultured , Culture Media/chemistry , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Lymphocytes/metabolism , Lymphocytes/pathology , Lymphocytes/ultrastructure , Mitochondria/enzymology , Mitochondria/radiation effects , Mitochondria/ultrastructure , Succinate Dehydrogenase/metabolism , Time FactorsABSTRACT
The effects of doxycycline on the morphology and function of donor erythrocytes were studied by scanning electron microscopy. The antibiotic in concentrations of 7.8 × 10(-5) and 7.8 × 10(-6) mol/liter induced heterogeneous changes in the surface architectonics in the erythrocyte population. These data indicated stimulation of erythrocyte "aging" under the effect of doxycycline.