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卷 88, 编号 8 (2023)
Articles
Superoxide anion radical generation in photosynthetic electron transport chain
摘要
The review analyzes the data available in the literature on the rates, characteristics and mechanisms of oxygen reduction to a superoxide anion radical at the sites of the photosynthetic electron transport chain where this reduction can occur. The existing assumptions about the role of the components of these sites in this process are critically examined, with use of thermodynamic approaches and the results of recent studies. The process of O2 reduction at the acceptor side of PSI, which is considered the main place of this process in the photosynthetic chain, is described in detail. The evolution of the photosynthetic apparatus in the context of controlling the leakage of electrons to O2 is considered. The reasons limiting the application of the results obtained with isolated segments of the photosynthetic chain to estimate the rates of O2 reduction at the corresponding sites in the intact thylakoid membrane are discussed.
Biohimiâ. 2023;88(8):1283-1301
1283-1301
Constitutive androstane receptor agonist initiates metabolic activity required for hepatocite proliferation
摘要
Constitutive androstane receptor (CAR, NR1I3) activation by chemical compounds evokes liver hyperplasia in rodent. 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a mouse CAR agonist, is most commonly used to study chemically induced liver hyperplasia and hepatocyte proliferation in vivo. TCPOBOP is potent murine liver chemical mitogen, which induces rapid direct liver hyperplasia independent of liver injury. In recent years, a lot of data has been accumulated on the transcription program that characterizes TCPOBOP-induced hepatocyte proliferation. However, there are scarce data about metabolic requirements of hepatocytes dividing upon treatment with xenobiotics. In present study, we employed liquid chromatography - mass spectrometry technology combined with statistical analysis to develop a metabolite profile of small biomolecules, to identify key metabolic changes in male mouse liver tissue after TCPOBOP administration. Analysis of biochemical pathways of the differentially affected metabolites in mouse livers demonstrated significant TCPOBOP-mediated enrichment of several processes including those relevant to nucleotide metabolism, amino acid metabolism, and energy substrate metabolism. Our findings provide evidence to support the conclusion that CAR agonist, TCPOBOP, initiates an intracellular program that promotes the global coordinated metabolic activities required for hepatocyte proliferation. Our metabolic data may provide novel insight into the biological mechanisms that occur during TCPOBOP-induced hepatocyte proliferation in mice.
Biohimiâ. 2023;88(8):1302-1312
1302-1312
The effect of hippocampal overexpression of dopamine neurotrophic factor (CDNF) on the behavior of mice with genetic predisposition to depressive-like behavior
摘要
Cerebral dopamine neurotrophic factor (CDNF) is the promising tool for treatment of Parkinson’s disease. However, its role in the regulation of non-motor behavior, including various psychopathologies, remains unclear. In this regard, the aim of present work was to study the effect of CDNF overexpression in the hippocampus on the behavior of ASC mice (Antidepressant Sensitive Cataleptics) with a genetic predisposition to depression-like behavior. CDNF overexpression in mouse hippocampal neurons was induced using an adeno-associated viral vector. Four weeks after stereotaxic injection of the AAV-CDNF construct into the dorsal hippocampus, home cage behavior, exploratory, anxiety and depressive-like behaviors, as well as spatial and associative learning were assessed. We found significant improvements in the dynamics of spatial learning in the Morris water maze in CDNF-overexpressing animals. At the same time, no effect of CDNF was found on other studied behaviors. The behavior of the experimental animals in the home cage conditions did not differ from that in the control group, except for a decrease in the total amount of food eaten and a slight increase in the number of sleep episodes in the light phase of the day. In the present study we also attempted to determine the molecular basis for the above mentioned changes by genes expression assessment. We did not find significant changes in the mRNA level of key kinases, genes involved in neuroplasticity and neuronal survival as well as genes encoding receptors for the main neurotransmitter systems. However, animals overexpressing CDNF show increased lever of spliced Xbp indicating activation of the Ire1α/Xbp1 pathway traditionally associated with ER stress. Immunohistochemical analysis showed that CDNF was co-localized with the ER marker calreticulin. Thus, the effects of endogenous CDNF on behavior that we have found may be mediated by a specific molecular cascade, which emphasizes its difference from classical neurotrophic factors.
Biohimiâ. 2023;88(8):1313-1336
1313-1336
SkQ1 improves immune status and normalises the activity of antioxidant and nadph-generating enzymes in adjuvant-induced rheumatoid arthritis in rats
摘要
Rheumatoid arthritis (RA) is a severe systemic autoimmune inflammatory disease. Oxidative stress and excessive formation of mitochondrial reactive oxygen species (ROS) are now considered to be the central pathogenetic mechanisms of connective tissue component destruction and the factors responsible for a highly active inflammatory process and autoimmune response. The aim of the present work was to evaluate the effect of mitochondrial-directed antioxidant 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on immune status, intensity of free radical-induced oxidation and functioning of antioxidant system (AOS) and NADPH-generating enzymes in rats with adjuvant-induced RA. Laboratory animals were divided into 4 groups: a control group; a group of animals with RA; animals injected intraperitoneally with SkQ1 at a dose of 1250 nmol/kg every 24 hours during the next 8 days from day 7 of RA development, and animals given SkQ1 at a dose of 625 nmol/kg according to the above scheme. Material for the study was taken on day 15 after the start of the experiment. The rate of erythrocyte sedimentation, the level of circulating immune complexes, and the concentration of immunoglobulins A, M and G were determined in rats by enzyme immunoassay. The intensity of free radical-induced oxidation was assessed by iron-induced biochemiluminescence, diene conjugate content and aconitate hydratase activity. Enzyme activity and metabolite content in animal tissues were analysed spectrophotometrically. The results of the work showed that the development of RA was associated with an increase in the indicators of immune response and intensity of free radical-induced oxidation. The development of an imbalance in AOS functioning and an increase in the activity of NADPH-generating enzymes was also observed. The administration of SkQ1 resulted in a dose-dependent change in the oxidative status indicators towards the control, which was accompanied by the normalization of the immune status parameters. It seems that the tested compound decreased the level of mitochondrial ROS, resulting in an inhibition of the inflammatory response. The consequence of these changes could be inhibition of free radical generation by immunocompetent cells and subsequent mitigation of the severity of oxidative stress in the tissues.
Biohimiâ. 2023;88(8):1337-1351
1337-1351
Analysis of transferrin in urine in patients with bladder cancer using nanobodies
摘要
It is known that the saturation ratio of transferrin (Tf) with iron in human blood is an important clinical parameter. Specific antibodies can be used to analyze subtle changes in the relative abundance of different forms of transferrin potentially associated with a pathological process. Recently, the authors of this study were able to obtain and characterize highly specific single-domain antibodies (nanobodies) that predominantly recognize the iron-saturated (holo-Tf) or iron-unsaturated (apo-Tf) form of transferrin. In this work, under conditions closer to physiological than in previous experiments, we further demonstrated that these unique nanobodies have extremely high differential binding specificity for different forms of Tf in different human biological fluids. Using these nanobodies, we were able to analyze for the first time the relative abundance of transferrin forms in urine samples from patients with bladder cancer (BC). We have shown that an increase in the concentration of total Tf in urine samples normalized for creatinine is associated with the degree of progress and growth of malignancy of BC. In the samples of healthy donors and in the early stages of BC (G1), Tf is detected in much smaller amounts (compared to later stages) and only with additional concentration of the studied samples. For most of the studied urine samples from BC patients, it is expected (as previously shown in the case of Tf in the blood of terminal ovarian cancer patients) that the concentration of apo-Tf is clearly higher than holo-Tf, especially in the case of the most advanced muscle-invasive BC. It was a surprise for us that approximately equal amounts of apo-Tf and “holo-Tf” were found in the urine samples of some patients with BC. We hypothesized that the “holo-Tf” fraction in this case can be largely represented by “secondary complexes” formed by apo-Tf in combination with ions other than Fe3+, which accumulate in the urine of some cancer patients and are able to bind to apo-Tf, changing its conformation towards holo-Tf. Using inductively coupled plasma ionization mass spectroscopy (ICP-MS), we obtained the first results confirming our hypothesis. The “holo-Tf” preparation in these urine samples was found to be highly enriched in zinc and nickel. Also in this preparation, a relative enrichment in cadmium is observed, but it is present in much lower concentrations. The data obtained indicate that the nanobody used, recognizing predominantly the iron-saturated form of transferrin (holo-Tf), is also capable of binding transferrin in association with other metal ions that are different from iron. This ability could potentially open up new possibilities in studies of the relative abundance of various metal ions in association with transferrin in human biological fluids in normal and pathological conditions.
Biohimiâ. 2023;88(8):1352-1365
1352-1365
Hybrid implants based on calcium-magnesium silicate ceramic diopside as a carrier of recombinant BMP-2 and demineralized bone matrix as a scaffold: ectopic osteogenesis in intramuscular implantation in mice
摘要
High efficiency of hybrid implants based on calcium-magnesium silicate ceramic diopside as a carrier of recombinant BMP-2 and xenogenic demineralized bone matrix (DBM) as a scaffold for bone tissue regeneration was demonstrated previously on the model of critical size cranial defects in mice. In order to investigate the possibility of using these implants for growing autologous bone tissue using the in vivo bioreactor principle in the patient’s own body, the effectiveness of the ectopic osteogenesis induced by them in intramuscular implantation in mice was studied. At 7 µg dose of BMP-2 per implant, after 1 week we observed a dense agglomeration of cells, probably skeletal muscle satellite precursor cells, with areas of intense chondrogenesis, the initial stage of indirect osteogenesis, around the implants. After 12 weeks, a dense bone capsule of trabecular structure was formed, covered with periosteum, with mature bone marrow located in the spaces between the trabeculae. The capsule volume was about 8-10 times the volume of the original implant. There were practically no signs of inflammation and foreign body reaction. Microcomputer tomography data showed a significant increase of the relative bone volume, number of trabeculae and bone tissue density in the group with BMP-2 in comparison with the group without BMP-2. Considering that DBM can be obtained in practically unlimited quantities, of the required size and shape, and the BMP-2 used, which is obtained by synthesis in E. coli cells, is relatively inexpensive, further development of the in vivo bioreactor model based on hybrid implants from BMP-2, diopside and xenogenic DBM seems promising.
Biohimiâ. 2023;88(8):1366-1377
1366-1377
Long-term experimental hyperglycemia does not impair macrovascular endothelial barrier integrity and function in vitro
摘要
Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.
Biohimiâ. 2023;88(8):1378-1391
1378-1391
Regulation of human dna primase-polymerase primpol
摘要
Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to replication blockage, DNA break formation, cell cycle disruption and apoptosis. Cells have adapted to DNA damage evolving mechanisms allowing to eliminate lesions prior to DNA replication (DNA repair) and mechanisms which help to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of pathways includes the mechanism of DNA synthesis restart at the damaged sites by DNA primase-polymerase PrimPol. Human PrimPol was first described in 2013. The properties and functions of PrimPol have been extensively studied in recent years, but very little is known about the regulation of PrimPol and the association of the enzyme dysfunction with diseases. The present review is devoted to an analysis of the mechanisms of human PrimPol regulation in the context of DNA replication. The interaction of PrimPol with other proteins is discussed in detail and an attempt is made to compile a possible pathway for the regulation of human PrimPol activity. The paper also addresses the relationship of PrimPol dysfunction with human diseases.
Biohimiâ. 2023;88(8):1392-1411
1392-1411
Protective effects of peroxiredoxin 6 in modeling proinflammatory response using RAW 264.7 macrophages
摘要
The aim of the work was to study the effects of peroxiredoxin 6 (PRDX6), a recombinant antioxidant protein, on the level of proinflammatory responses caused by endotoxin exposure to RAW 264.7 macrophages. The addition of LPS to the RAW 264.7 cell culture medium expectedly increased the production of TNF-α, and the addition of PRDX6 led to a significant decrease in its production by 15-20%. The level of production of another proinflammatory cytokine, IL-1β, significantly activated by endotoxin, was completely normalized under the PRDX6 action. In addition, the addition of PRDX6 reduced the production of reactive oxygen species (ROS) induced by endotoxin and also prevented overexpression of the iNos gene in RAW 264.7 cells. The results showed that PRDX6 has a suppressive effect on the expression of the Nfe2l2 gene and the production of the transcription factor NRF-2 during the first 6 h of cell culture. The addition of endotoxin causes the activation of the NF-κB and SAPK/JNK signaling cascades, while in the presence of PRDX6, the activity of these signaling cascades decreases. It is known that the proinflammatory response of cells caused by bacterial LPS leads to the activation of apoptosis and the elimination of damaged cells. Our studies confirm this, since LPS leads to the activation of the Trp53 gene, a marker of apoptosis. The addition of peroxiredoxin 6 in the first hours of the development of an acute proinflammatory response leads to the suppression of Trp53 gene expression, which indicates a protective effect of PRDX6 that reduces apoptosis in RAW 264.7 macrophages.
Biohimiâ. 2023;88(8):1412-1422
1412-1422
Complexes and supramolecular associates of dodecyl-containing oligonucleotides with serum albumin
摘要
Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with a high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins via electrophoretic mobility shift assay. The results testify to the formation of albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that interaction of human serum albumin with a duplex of complementary dodecyl-containing oligonucleotides resulted in associates of round shape with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and with polydeoxyadenylic acid and dodecyl-containing oligothymidylate also resulted in formation of supramolecular associates with a size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm. The obtained data allow considering dodecyl-containing oligonucleotides and albumin as potential components for the development of methods to design self-assembled systems for solving problems of molecular biology, biomedicine, and the development of unique theranostics with targeted action.
Biohimiâ. 2023;88(8):1423-1440
1423-1440