Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.3, #4)

Prediction of functionally related proteins by comparative genomics in silico by M. A. Pyatnitskiy; A. V. Lisitsa; A. I. Archakov (323-334).
The review considers the computational prediction of functionally related proteins by comparative genomics. Growing possibilities of biotechnology for genome sequencing lead to generation of sequences for millions of genes. However, functions of majority of these genes remain unknown, and can be determined experimentally only for a few of them. Therefore, accurate and robust methods for in silico prediction (annotation) of gene functions are needed. We describe here the main techniques of comparative genomics, including the standard method based on transferring functions between homologous sequences and also context-based methods, including phylogenetic profiles and gene-neighbor approaches. Modern methods of comparative genomics allow obtaining correct functional annotations for more than a half of all organism proteins.
Keywords: related proteins; protein-protein interactions; functional annotation; phylogenetic profiles; comparative genomics

Non-enzymatic glycation of proteins: From diabetes to cancer by N. A. Ansari; Z. Rasheed (335-342).
Incubation of proteins with glucose leads to their non-enzymatic glycation and formation of Amadori products known as an early glycation product. Oxidative cleavage of Amadori products is considered as a major route to advanced glycation endproducts (AGEs) formation in vivo. Non-enzymatic glycation of proteins or Maillard reaction is increased in diabetes mellitus due to hyperglycemia and leads to several complications such as blindness, heart disease, nerve damage, and kidney failure. The early and advanced glycation products are accumulated in plasma and tissues of diabetic patients and cause production of autoantibodies against corresponding products. The advanced glycation products are also associated with other diseases like cancer. This review summarizes current knowledge of these stage specific glycated products as common and early diagnostic biomarkers for the associated diseases and the complications with the aim of a novel therapeutic target for the diseases.
Keywords: Non-enzymatic glycation; Amadori product; advanced glycation endproducts (AGEs); hyperglycemia; diabetes mellitus; cancer

Zebrafish as a model system for biomedical studies by N. F. Belyaeva; V. N. Kashirtseva; N. V. Medvedeva; Yu. Yu. Khudoklinova; O. M. Ipatova; A. I. Archakov (343-350).
Zebrafish (Danio rerio) is now firmly recognized as a powerful research model for many areas of biology and medicine. Here, we review some achievements of zebrafish-based assays for modeling human diseases and for drug discovery and development. For drug discovery, zebrafish is especially valuable during the earlier stages of research as its represents a model organism to demonstrate a new treatment’s efficacy and toxicity before more costly mammalian models are used. This review considers some examples of known compounds which exhibit both physiological activity and toxicity in humans and zebrafish. The major advantages of zebrafish embryos consist in their permeability to small molecules added to their incubation medium and chorion transparency that enables the easy observation of the development. Assay of acute toxicity (LC50 estimation) in embryos can also include the screening for developmental disorders as an indicator of teratogenic effects. We have used the zebrafish model for toxicity testing of new drugs based on phospholipid nanoparticles (e.g. doxorubicin). Genome organization and the pathways involved into control of signal transduction appear to be highly conserved between zebrafish and humans and therefore zebrafish may be used for modeling of human diseases. The review provides some examples of zebrafish application in this field.
Keywords: zebrafish (Danio rerio); drug discovery; acute toxicity; teratogenesis; apoptosis; cancer

During the last years the low-molecular non-peptidic regulators of the polypeptide hormone receptors containing LGR-repeats (LGR-receptors) were identified. The review summarizes and systematizes data on structure and molecular mechanisms realizing the effects of such regulators as agonists and antagonists of the luteinizing, follicle-stimulating and thyroid-stimulating hormones. The regulators interact with the serpentine domain of LGR-receptors and trigger the receptor-coupled signaling cascades. Low-molecular agonists and antagonists of the LGR-receptors are considered as a new generation of drugs that can demonstrate highly sensitive and selective regulation of the functional activity of signaling systems sensitive to pituitary glycoprotein hormones. These regulators are more available than these hormones and can be used orally.
Keywords: luteinizing hormone; low-molecular agonist; receptor; thyroid stimulating hormone; follicle-stimulating hormone; LGR-repeat

Mass spectrometry identification of cytochrome P450 2B4 interaction sites for NADPH: Cytochrome P450 reductase by A. V. Ivanov; A. T. Kopylov; V. G. Zgoda; I. Yu. Toropygin; E. V. Khryapova; Yu. D. Ivanov (361-371).
The interaction sites for protein partners, cytochrome P450 2B4 (d-2B4) and NADPH: cytochrome P450 reductase (d-Fp), have been identified. These proteins form complexes during their functioning. Nonspecific covalent cross-linking of the d-2B4 complexes with d-Fp in the Emulgen 913 monomerized system was achieved by 4,4′- dithiobis-phenyl azide. Covalently cross-linked peptides of this complex were identified by ESI-MS/MS. Several binding sites have been identified for these proteins. Based on these sites a model for intermolecular interaction between these proteins has been proposed. This model includes 5 contact sites on d-2B4 for d-Fp (stabilized by the cross-linker); these include the following pairs of corresponding peptides of d-2B4 and d-Fp: 1) d-2B4324–336 and d-Fp570–585; 2) d-2B4423–433 and d-Fp102–109; 3) d-2B4327–336 and d Fp452–464; 4) d-2B4192–197 and d-Fp456–464; 5) d-2B4134–139 and d-Fp406–425. In the two last cases d-Fp peptides are located in the interdomain cleft and stabilize the protein-protein complex via the cross-linker and so the d 2B4/d-Fp complex formation by these sites may involve amino acid residues of the peptides d-Fp456–464 and d-Fp406–425, which surround the interdomain cleft.
Keywords: cytochrome P450 2B4; NADPH: cytochrome P450 oxidoreductase; mass spectrometry; dithiobisphenyl azide; interaction sites

The level of glycemia, contents of free radical oxidation products (thiobarbituric acid reactive substances, oxidatively modified proteins) have been investigated in blood plasma and heart of rats with diabetes mellitus subjected to chronic alcohol intoxication. Preexisting diabetes mellitus had no influence on the effect of chronic alcohol consumption on the blood plasma levels of oxidatively modified proteins, thiobarbituric acid reactive substances and glucose. However, the contents of thiobarbituric acid reactive substances and products of oxidative modification of proteins were significantly higher in hearts of diabetic rats with chronic alcohol intoxication than in diabetic rats without alcohol intoxication or in rats subjected to chronic alcohol treatment. The alcohol-induced hyperactivation of free radical processes found in the heart may have additional damaging effect.
Keywords: diabetes mellitus; chronic alcohol intoxication; thiobarbituric acid reactive substances; oxidatively modified proteins

The activity of liver microsomal detoxication enzymes has been investigated in tumor-bearing rats exposed to preliminary irradiation. Preliminary irradiation of rats (before transplantation of Guerin’s carcinoma) resulted in the decrease of liver microsomal cytochrome ê450 and glutathione-S-transferase observed in the latent and logarithmic phases of oncogenesis as compared with the non-irradiated tumor-bearing rats. The decrease in cytochrome P450 activity may be associated with its transition into an inactive form, cytochrome P420. During subsequent steady state period the tumor development is the factor determining changes of the studied parameters because the activity of phase I and II detoxification components tends to the level found in tumor-bearing rats, which were not exposed to pilot irradiation.
Keywords: cytochrome P450; glutathione-S-transferase; microsomal fraction; liver; Guerin’s carcinoma

Activity of key enzymes of the sphingomyelin cycle and the content of its components (sphingomyelin, ceramide and sphingosine-1-phosphate) have been studied in livers of rats in dynamics of acute toxic hepatitis induced by subcutaneous administration of oil solution of CCl4. Sphingomyelinase activity significantly increased already on early terms and remained increased over the whole period of observation. Ceramidase activity insignificantly differed from the control level. The levels of sphingomyelin and sphingosine-1-phosphate did not undergo marked changes while ceramide content significantly increased. The balance between liver content of ceramide (proapoptotic) and sphingosine-1-phosphate (the antiapoptotic factor) was shifted towards ceramide over the whole observation period. In sphingomyelin molecules there was a significant decrease in the content of the fatty acids C18:1 and C22:2, while in ceramide molecules and sphingosine-1-phosphate only the fatty acid C22:2 changed. In spite of a significant decrease in the content of some unsaturated fatty acids, calculated unsaturation coefficients of the fatty acid component of the sphingomyelin cycle metabolites insignificantly differed from control.Taking into consideration literature data our results suggest involvement of ceramide-mediated apoptosis in the pathogenesis of acute toxic hepatitis. Elimination of damaged hepatocytes permits realization of repair processes and promotes optimization of cellular community in the liver.
Keywords: acute toxic hepatitis; sphingomyelin cycle

The effect of complexes of precursors and modulators of coenzyme Q biosynthesis on the functional state of heart mitochondria of old rats by E. B. Kuchmenko; D. N. Petukhov; G. V. Donchenko; L. S. Mkhitaryan; S. V. Tymoshchuk; N. A. Strutynskaya; G. L. Vavilova; V. F. Sagach (386-392).
Aging is accompanied by changes in activity of electron-transport enzyme complexes in myocardial mitochondria of old rats and by increased sensitivity of the mitochondrial permeability transition pore (MPTP) to inductors of its opening (Ca2+ and phenylarsine oxide). We also observed activation of lipid and protein free-radical peroxidation processes. Administration of a complex of biologically active substances that included precursors and modulators of coenzyme Q biosynthesis (α-tocopherol acetate, 4-hydroxybenzoic acid, and methionine) caused the increase in coenzyme Q content, correction of functional activity of mitochondrial electron-transport chain enzyme complexes, the decrease in intensity of lipid and protein free-radical peroxidation in the heart mitochondria and the decrease in sensitivity of mitochondrial permeability transition pore to inductors of its opening. This complex may be recommended for treatment of mitochondrial dysfunction in various pathologies of cardiovascular system, including in aging.
Keywords: ubiquinone; old animals; lipid and protein free-radical peroxidation; mitochondrial permeability transition pore

Cathepsin K and matrix metalloprotease activities in bone tissue of the OXYS rats during the development of osteoporosis by A. A. Venediktova; O. V. Falameeva; N. G. Kolosova; M. A. Sadovoj; T. A. Korolenko (393-398).
The activity of osteoclast-specific cysteine protease, cathepsin K, and matrix metalloproteases (MMPs) has been investigated in bone tissue of senescence-accelerated OXYS rats and in Wistar rats. At the age of 3 month (the period preceding manifestation of osteoporosis in OXYS rats) cathepsin K activity was higher whereas MMP activity was lower in Wistar rats. At the age of 14 months Wistar rats cathepsin K activity increased and MMP activity decreased. The age-related changes in bone cathepsin K and MMP activity of OXYS rats had opposite direction. Thus, despite of marked manifestations of osteoporosis previously found by us in OXYS rats (the decrease in mineralization density of the bone tissue and its resorption) no interstrain differences in cathepsin K and MMPs were found between Wistar and OXYS rats. Activity of a universal protease inhibitor, α2-macroglobulin, was higher in serum of 14-month old OXYS rats than in Wistar rats of the same age. The role of cathepsin K activation in resorption of bone tissue in the development of osteoporosis in senescence-accelerated OXYS rats is discussed.
Keywords: cathepsin K; matrix metalloproteases; bone resorption; early ageing OXYS rats; osteoporosis

Impairments in the serum redox thiol disulfide system have been investigated in HIV carriers and in patients with clinical manifestations of AIDS. The role of its components (total, protein and non-protein-SH, -S-S- groups) in the mechanism responsible for formation of oxidative stress and distress in HIV is substantiated. The clinical and laboratory criterions of manifested peroxidation processes in HIV infection are specified in terms of blood serum parameters reflecting impairments in redox transformations of -SH, -S-S- groups, neutralization and utilization of MDA and stability of LPC. Taking into consideration a correlation between impairments in the parameters studied, oxidative stress and distress and clinical manifestations of HIV infection we refer this pathology to “free radical diseases”. In this connection, the parameters studied may be used as additional biochemical markers of oxidative stress and distress, and also for prescription of antioxidants and their combinations in complex prophylactic or therapeutic application in HIV infection.
Keywords: thiol disulfidic system; oxidative stress and distress; HIV infection; AIDS

Measurement of endogenous alloxan in human blood by D. A. Korzhenevskiy; A. A. Selischeva; S. V. Saveliev (404-407).
For determination of endogenous alloxan content in blood of healthy donors it was stabilized by rapid lowering pH in a sample to 2.0. Alloxan was then allowed to form a colored product in a reaction with [ital]o-phenylenediamine and its content was measured by spectrophotometry using an internal calibration curve. Analysis of 75 blood samples of healthy volunteers has shown that in most donors alloxan concentrations vary from 41 to 265 µmol/l. However, in a small group of healthy people alloxan levels greatly exceeded the above-mentioned limit.
Keywords: blood; alloxan; spectrophotometry