The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻⁶ M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻⁸ M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.
Regulatory T-cells CD4⁺CD25⁺FoxP3⁺CD127low (Tregs) play a key role in the maintenance of tolerance to auto antigens, inhibit function of effector T and B lymphocytes, and provide a balance between effector and regulatory arms of immunity. Patients with autoimmune diseases have decreased Treg numbers and impaired suppressive activity. Transformed ex vivo autologous Tregs could restore destroyed balance of the immune system. We developed a method for Treg precursor cell cultivation. Following the method, we were able to grown up 300-400 million of Tregs cells from 50 ml of peripheral blood during a week. Transformed ex vivo Tregs are 90-95% CD4⁺CD25⁺FoxP3⁺CD127low and have increased expression of transcription genes FoxP3 and Helios. Transformed ex vivo Tregs have increased demethylation of FoxP3 promoter and activated genes of proliferation markers Cycline B1, Ki67 and LGALS 1. Transformed ex vivo Tregs have increased suppressive activity and up to 80-90% these cells secrete cytokines TNFα и IFNγ. Our data suggest transformed ex vivo autologous Tregs have genetic, immunophenotypic and functional characteristics for regulatory T-cells and further can be used for adoptive immunotherapy autoimmune diseases and inhibition of transplantation immunity.
Using a model of the human SK-Mel-147 melanoma cell line, it was shown that blocking the expression of integrin α3β1 by transduction of cells with α3-specific shRNA did not affect their proliferation, but sharply increased the proportion of SA-β-Gal-positive cells, a phenotypic feature of cell senescence. These findings were accompanied by a significant increase in the activity of the Akt and mTOR protein kinases and the expression of p53 and p21 oncosupressors. Pharmacological inhibition of mTORC1 reduced the number SA-β-Gal-positive cells in the SK-Mel-147 cell population depleted of α3β1. Based on our recent data on a non-canonical function of Akt isomers in the regulation of SK-Mel-147 cell senescence caused by deficiency of α2β1 receptor, we investigated the role of Akt isomers in senescence induced by the α3β1 knockdown. It appeared that in the cell population with downregulated α3β1, inhibition of Akt1 reduced the number SA-β-Gal positive cells to the level of control cell population, while inhibition of Akt2 had no visible effect. Our results demonstrate that the laminin-specific integrin α3β1, like the collagen-specific receptor α2β1, is involved in tumor cell protection from senescence, and senescence induced by α3β1 depletion, like that caused by α2β1 deficiency, is based on a signaling mechanism employing a non-canonical function of the Akt1 isoform.
Bioprosthetic heart valves (BHVs) have lower thrombogenicity rates and excellent hemodynamic parameters similar to native valves. However, the lifespan of these medical devices is limited to 15 years due to the structural valve degeneration (SVD). One of the mechanisms underlying functional impairment and calcification of BHVs includes proteolytic degradation of biomaterials. However, proteases found in xenogeneic tissue of BHVs remain poorly studied. In this study using the dot blot assay, we have performed a screening analysis of proteolytic enzymes and their inhibitors in the leaflets of five BHVs explanted due to dysfunction. Five aortic valves (AVs) explanted due to calcific aortic valve disease were used as a comparison group. The results of the study have demonstrated the presence of at least 17 proteases and 19 of their inhibitors in BHVs. In the AVs 20 proteases and 21 of their inhibitors were identified. Small quantitative differences were found between proteomic profiles of BHVs and AVs. Matrix metalloproteinases (MMPs) were expressed in BHVs and AVs at comparable levels, but the level of tissue inhibitors of metalloproteinases-1/-2 and reversion-inducing-cysteine-rich protein with Kazal motifs in implant tissues was lower than in native valves. This suggests that excessive activity of MMPs cannot be counterbalanced by their specific inhibitors in BHVs and therefore MMPs initiate the process of degeneration. Moreover, the detection of a wide range of proteolytic enzymes and their inhibitors in the degenerated BHVs suggests the existence of several pathophysiological pathways that can lead to SVD.
A significant decrease in the activity of liver lactate dehydrogenase (LDH, EC 1.1.1.27) associated with a decrease in the expression of the corresponding genes was found in rats with alloxan-induced diabetes. The decrease in the LDH activity was due to the cytoplasmic isoform of this enzyme. It was found that the level of ldha and ldhb gene transcripts in the liver of healthy rats was higher than in animals with alloxan diabetes. The ldha gene expression demonstrated almost 9-fold decrease, while a decrease in the ldhb gene expression was less pronounced (just 1.25-fold). Our data indicate an important role of LDH in the adaptive response of cellular metabolism in the development of type I diabetes mellitus.
Sialic acids (SA) are neuraminic acid derivatives, located at the terminal position in the chains of monosaccharide residues of various glycoconjugates. SA play a dual role: they either mask recognition sites, or, on the contrary, represent biological targets that can be recognized by receptor proteins and serve as ligands. The desialylation/sialylation processes can be considered as a dynamic modification regulated by sialyltransferases and sialidases in response to external or internal stimuli. This review describes the structural and functional diversity and the potential use of SA fractions as biomarkers for various pathological conditions. Almost any extreme impact on the body and inflammatory processes are accompanied by an increase in the level of both total and free SA in the blood and tissues. Possible reasons for the increase of sialoglycoconjugate metabolism indicators in biological material include: (i) activation of the hepatocyte synthesis and secretion of various acute-phase proteins, many of which are sialoglycoproteins, (ii) impaired membrane integrity and destruction of body cells, (iii) high activity of sialidases (neurominidases) and sialyltransferases. Most acute and chronic liver diseases are characterized by the decrease in the total level of SA in the blood serum (because many plasma proteins are synthesized and glycosylated in hepatocytes). Aberrant sialylation results in changes of sialoglycoconjugate structure, its ability to perform biological functions and sialoglycoconjugate half-life. Glycosylation is the most common post-translational modification of proteins in the virus, which not only promotes the formation of specific conformation of viral proteins, but also modulates their interaction with receptors and affects host cell recognition, viral replication and infectivity. Serum total SA concentration increases in some benign and inflammatory conditions, which indicates a lack of specificity and limits their use for early detection and screening of neoplastic diseases. Clinical and diagnostic value of determining the sialoglycoconjugate metabolic indicators, including changes in the content of both SA fractions and specific proteins in various biological fluids and tissues, consists in establishing the causes and mechanisms of biochemical changes in the body in certain diseases. In combination with the measurement of existing markers, they can be used to improve diagnosis, staging and monitoring of therapeutic response in some pathological conditions where the need for specificity is less than for specific diagnostics.
The fractions of 26S and 20S proteasomes have been isolated from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to the proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes involved in various metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.
In order to create new oral vanadyl organic complexes-based drugs for the treatment of diabetes mellitus biligand vanadyl derivative of L-malic acid (bis(L-malato)oxovanadium(IV) was prepared and its potential as a novel hypoglycemic agent was studied in the streptozotocin-diabetic rats. We show that the oral administration of bis(L-malato)oxovanadium(IV) with drink water significantly reduced glucose concentration in blood and urine, as well as the level of glycated proteins in the streptozotocin-diabetic rats.
Functional activity of glycolysis was studied in liver and skeletal muscles of rats subjected to acute alcohol intoxication. Alcohol administration in the dose of 1 g/kg led to inhibition of glycolytic enzymes activities and decreased levels of some glycolytic substrates in liver. Activity of LDH increased in skeletal muscle under these experimental conditions. Moderate dose of ethanol (2.5 g/kg) activated glucogenolysis and increased glucose level in liver. In this group inhibition of PFK activity and decrease of glucose-6-phosphate level in muscle was also observed. Alcohol administration in the dose of 5 g/kg caused inhibition of rate-limiting enzymes of glycolysis in liver and muscle.
The effect of ozonated physiological solution on lipid composition, lipid peroxidation and level of carbohydrate metabolism substrates were investigated in the early reperfusion period. The total ischemia/reperfusion model was used. This study shows that injection of ozonated physiological solution in the early reperfusion period did not prevent cardiac myocyte membrane delipidization, activation of lipid peroxidation due to antioxidation exhaustion. Treatment with ozonated physiological solution promotes normalization in the lysophosphatidylholine and lysophosphatidylserine content, activation of hydrolytic degradation of neutral lipids, the decrease in membrane lipid microviscosity, activation of the aerobic glucose utilization and prevents lactic acidosis in the heart.
The membrane, antioxidant and functional effects of vinpocetine and a-tocopherol have been investigated under conditions of acute experimental cerebral ischemia in rats. Vinpocetine administration decreased accumulation of lysophospholipids in brain plasma membranes. Vinpocetine also blocked accumulation of conjugated dienes (CD). alpha-Tocopherol inhibited augmentation in CD content and did not reduce the level of lysophospholipids in brain plasma membranes. Functional consequences of membrane impairments were also detected in some behavioral tests and physical capabilities. Administration of both vinpocetine and alpha-tocopherol decreased manifestations of the altered parameters induced by cerebral ischemia and vinpocetine was more effective than alpha-tocopherol.
The experimental data indicate that melatonin actively influences time-causes of changes of lipid content in rats tissue during the inflammation process. Its effect depends on a dose, modes of administration (intraperitonial, hypodermic or local) and duration of treatment. A single dose intraperitonial administration of melatonin (4 mg/kg) did not influence lipid content in the granular-fibrose tissue, while repeated injections of this hormone limited the increase in contents of lipids and phospholipids at the 5th and 8th days of regeneration. Long-term subcutaneous injections of melatonin caused distinct changes of lipids: at the dose of 0.3 mg/kg it prevented, and at the dose of 4 mg/kg it promoted the increase of lipid content in the granular-fibrose tissue. Local application of a melatonin solution (1.5 mg/ml) at early periods of regeneration caused insignificant changes of total lipids and total phospholipids in the granular-fibrose tissue. However, the higher concentration (15 mg/ml) of melatonin caused the decrease of total lipids due to reduced content of cholesterol and triglycerides and the increase of total phospholipids and some of their fractions.
Using recombinant microorganisms S. cerevisiae GRF18/YEp 5117α, expressing bovine adrenocortical cytochrome P450cl7, we have studied the effect of various modifiers of steroid biosynthesis on the relationship between reactions of the 17α-hydroxylation and 20α-reduction of progesterone. Dexamethasone and metyrapone had no effect on the reaction of progesterone 17α-hydroxylation and 20α-reduction of 17α-hydroxyprogesterone. Mifepriston and danazol did not covalently modify amino acid residues of the cytochrome P450cl7 or its heme group under the conditions of progesterone biotransformation by recombinant yeasts. Ketokonazole, mifepriston and danazol were found to be low-affinity competitive inhibitors, but the 20-dihydroderivatives of progesterone were mixed type inhibitors of the cytochrome P450cl7. All modifiers used did not affect the functional properties of the yeast analog of 20α-hydroxysteroid dehydrogenase. Based on the effect on catalytic parameters of the cytochrome P450cl7, the all modifiers used can be arranged in the following order: 20β-dihydroprogesterone (maximal effect) > mifepriston = ketokonazole > 20α-dihydroprogesterone > danazol > dexamethasone, metyrapone (without effect).
Dramatic improvement in computer performance is observed during the last decades. In molecular modeling field this allows more and more complex tasks to be tackled by means of quantum chemistry available for a broader research community, including researchers, whose primary scientific interests are far from molecular modeling. At the same time, user interface of many molecular modeling programs is oriented on professional users with strong computer science background. The WebQC newly developed web-interface is intended to cater needs of newcomer users of molecular modeling programs and quantum chemistry methods. The software provides uniform interface to various molecular modeling packages. 3-D visualization of molecular geometry configuration is provided as well. Currently, the PC GAMES and Dalton quantum chemistry programs were integrated with the WebQC, but the integration of new packages requires only modification of WebQC configuration files, which are of XML format. The software can be helpful in providing remote access to computing facilities, in education and facilitate teamwork of geographically distributed research groups
The amino acid sequence of porcine beta-lipotropin was the first protein primary structure studied in Russia. This peptide as well as ACTH is liberated after proteolysis of proopiomelanocortin (POMC). alpha-MSH and beta-MSH (melanocortins), which are the fragments of ACTH and beta-lipotropin respectively, are the mediators of leptin action on appetite and lipid metabolism. The structure and molecular aspects of hormone signaling of the membrane receptors of leptin and melanocortins were analysed in the connection to the regulation of food consumption, growth, and puberty. Some aspects of insulin receptor and IGF-I receptor as well as intracellular receptors of lipid hormones (steroid and thyroid hormones) were also discussed. The postulate: "All organs, tissues, and cells of humans and animals are endocrine" is formulated on the basis of the accumulated data.
Both metronidazole and aminotriazole increased while sanazole (drug AK-2123) decreased the NADPH/lucigenin-dependent chemiluminescence of liver microsomes of phenobarbital-treated rats. Sanazole strongly inhibited the lucigenin-dependent chemiluminescence in the enzyme system of xanthine-xanthine oxidase. Aminotriazole and metronidazole were less potent inhibitors of chemiluminescence less than sanazole. All these azole derivatives did not absorb light in the region of light emission of lucigenin. Both lucigenin and sanazole increased the rate of cytochrome c reduction by microsomes in case of using NADPH as a donor of electrons, whereas no effect of metronidazole and aminotriazole on this rate was found. The sanazole inhibition of lucigenin-dependent chemiluminescence could reflect competition between sanazole and lucigenin for electrons in the active centre of flavin reductases. Thus, microsomal NAD(P)H-reductases can be potentially involved in a bioactivation of sanazole. Lucigenin-dependent chemiluminescence cannot be used for measuring the modulating action of agents on reactive oxygen species production in the microsomes, but it may be used for luminometrical studies of enzyme complex NAD(P)H-reductases/cytochrome P450 in model systems.
Information on the complete genome sequences of a number of organisms available recently offers essentially new opportunities for the development of new, highly effective antimicrobial compounds. In particular, the search for new effective antituberculosis drugs remains an important problem, due to the recent increase of number of patients suffering with tuberculosis. In this respect considerable attention is paid to the cyp51-like gene Rv0764c encoding sterol-14 alpha-demethylase belonging to the cytochrome P450 superfamily, which has been discovered by computer analysis of the Mycobacterium tuberculosis genome sequence. We have screened 64 clinical isolates of M. tuberculosis for functionally relevant mutations in the coding sequence of the gene encoding Cyp 51-demethylase by single-strand conformation polymorphism analysis (SSCP) and sequencing of PCR-amplified gene fragments. Structural analysis of the gene in the isolates revealed no mutations leading to amino acid substitutions in the corresponding protein. 10 isolates had a silent nucleotide substitution 114 GCT-->GCC. Computer analysis of cyp51 sequence of the CDC1551 strain also revealed a similar nucleotide substitution, which has not been mentioned previously. The data obtained demonstrate that the sequence of the gene is highly conserved, supporting the advisability of M. tuberculosis Cyp51 protein to be considered as a molecular target for new antitubercular drugs. The SNP found in the gene coding sequence may be employed in the studies of M. tuberculosis population genetics.
The data on biochemical and molecular-genetic diagnostics of a hereditary lisosomal storage disease, late infantile neuronal ceroid lipofuscinosis (CLN2) are presented. The disease is associated with a hereditary deficiency of pepstatin-unsensitive peptidase--tripeptidylpeptidase 1 (TPP1)--caused by mutations in the TPP1-coding gene CLN2. Among the 30 patients with clinical manifestations of CLN, six patients with a pronounced decrease in TPP1 activity were revealed; these data were interpreted as indicating the presence of CLN2 in these patients. The analysis of the isolated DNA indicated the availability of the most widespread mutation g3670 C > T(R208X) leading to the untimely termination of TPP1 synthesis. It was shown that in 5 patients this mutation is present in homozygous state and in one patient, in the heterozygous state. In this patient a hitherto unknown mutation, g3665G > A (R206H), was revealed. The pathogenetic significance of this mutation and the importance of molecular-genetic diagnosis of CLN are discussed with regard to medico-genetic consulting and prenatal diagnosis of this disease.
The review highlights current aspects of a large group of diseases the main pathogenetic element of which is an inherited or acquired disturbance of gene expression of nuclear or mitochondrial genome encoding mitochondrial proteins. The recent data on mutant genetic loci specific to the most wide spread mitochondrial diseases are considered. The steps of pathogenesis, include the mutations of nuclear or mitochondrial genes, disturbances of mitochondrial protein synthesis, dissipation of proton membrane potential, opening of a permeability transition pore, releasing of procaspases, cytochrome c, and other proapoptotic molecules, and finally chromatin fragmentation and apoptotic cell death. We discuss the possible reasons of polysymptomatic character and different variants of mitochondrial disease manifestations on the basis of the phenomenon of mitochondrial DNA heteroplasmy and metabolic compensation of the genetic defects. Modern biochemical methods of a mitochondrial disease diagnostics: (PCR-amplification, polarographic research of mitochondrial respiration and oxidative phosphorylation, analysis and monitoring of metabolites in biological liquids) are characterized. The basic principles and perspectives of the treatment of mitochondrial diseases, (gene therapy, correction of metabolic disorders, application of antioxidants and neuropeptides) are described.
