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.
Abstract Using computer modelling, 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 «anchors» to the surface.
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 Fe
3
O
4
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 Fe
3
O
4
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.
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 10 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.
Fructan-modifying enzymes are divided into fructan-producing enzymes (fructosyl transferases) and fructan hydrolyzing enzymes (invertases, inulinases, levanases). Fructosyl transferases break the ...glycosidic bond of sucrose and use the energy of this bond to attach the resulting fructosyl to another sucrose molecule or other acceptor, increasing the fructan chain. Invertases hydrolyze sucrose and small fructooligosaccharides. Oligo- and polyfructans are cleaved by inulinases and levanases. A difference of only three amino acid residues affects the ability of glycoside hydrolases to cleave various substrates, in particular inulin and levan, or to exhibit transfructosylating activity. In this regard, the aim of the work was to carry out a comparative analysis of the primary structures of glycoside hydrolases of various origins. The paper presents the results of a comparative analysis of the amino acid sequences of glycoside hydrolases from the NCBI database (
https://www.ncbi.nlm.nih.gov/
). The overlap percentage (Query cover) of the sequences and their identity (Ident) were calculated using the Blast program (
https://blast.ncbi.nlm.nih.gov/Blast.cgi
). It was found that the affinity of endoinulinase from
Aspergillus ficuum
with 6- and 1-fructan exohydrolases from
Arabidopsis thaliana
and
Arabidopsis lyrata
subsp. Lyrata was higher (89% overlap and 24% identity) than exoinulinase from
Kluyveromyces marxianus
(38 and 57% overlap, 29 and 26% identity, respectively). Fructan 1-exohydrolase I from
Cichorium intybus
was also closer in primary structure to fungal endoinulinase (90% overlap and 25% identity) than to yeast exoinulinase (51% overlap and 27% identity). From the results obtained, the following conclusion can be drawn: the mechanism of substrate hydrolysis does not in all cases determine the degree of homology of glycoside hydrolases and related enzymes. It is possible that some glycoside hydrolases, including inulinases, can act both as endo and exo-enzymes, i.e., possess both types of catalytic activity towards fructans.
The effect of UV-light (240-390 nm) in doses of 151 and 755 J/m
2
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.
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/m
2
. 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 р53, cytochrome
с
, 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/m
2
involves the р53-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.
An analysis of the effect of UV radiation on human lymphocytes and keratinocytes is presented. The main effects of UV irradiation are related to changes in signal transmission or the functioning of ...signaling pathways in cells. As a result, a number of intracellular processes change (protein synthesis, cell metabolism, triggering programmed or nonprogrammed cell death, etc.). This article discusses the influence of UV irradiation on cell metabolism, on the synthesis of several proteins (including transcription regulation by active forms of oxygen), on the concentration of calcium in the cell and the calcium-dependent regulatory path, on the receptor profile, and on different ways of cell death. Currently, there is no unified scheme describing possible ways to realize the energy of UV radiation in different types of cells. Based on our own experimental data and literature analysis, we have proposed a scheme that includes the most likely events in UV modified lymphocytes during their incubation. When using the same radiation dose, depending on the state of cells and the incubation conditions, different scenarios are possible: death by apoptosis or necrosis or an increase in the functional activity of cells. The need to study the mechanisms of implementation of UV-induced cellular response is due to both the increase in the intensity of UV radiation in the atmosphere and the prospects for the use of phototherapy.
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.
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.
