Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.5, #2)

Antimicrobial peptides: the mode of action and perspectives of practical application by S. A. Okorochenkov; G. A. Zheltukhina; V. E. Nebol’sin (95-102).
This review deals with antimicrobial peptides (AMPs) that demonstrate antibacterial, antiviral, and antifungal activity. It considers structure and mechanism of AMP interaction with lipid membrane and intracellular targets of pathogens. Special attention is paid to modern state and perspectives of AMP practical application and also to approaches that increase efficacy and reduce toxicity of AMP by chemical modification of their structure.
Keywords: antimicrobial peptides; mode of action; practical application

Stress and the thyroid gland by L. I. Nadolnik (103-112).
The review highlights the effects of acute and chronic stress on thyroid gland metabolism. Special attention is paid to the influence of stress and the direct effects of glucocorticoids on the thyroid status, the activities of thyrocytes, iodine uptake, its oxidation and organification as well as peripheral metabolism of thyroid hormones (deposition and transport of thyroid hormones, deiodinase activities in different tissues). The role of stress in the development of thyroid pathology is analyzed and characteristic features of thyroid function alterations during impaired functioning of the pituitary-adrenal system are established. Taking into consideration serious consequences of thyroid deficiency for the body, even in subclinical thyroid insufficiency, the mechanisms of the stress-induced impairments in thyroid functions are of interest for further studies.
Keywords: stress; glucocorticoids; thyroid gland; thyroid hormones; iodine absorption and organification; deiodinases

Mechanisms of the anticancer effects of polyphenols, found in fruits, vegetables, spices and representing parts of daily nutrition, have been considered. These compounds may be the basis for the development of cancer preventive preparations. They can block initiation of carcinogenesis by inactivating exogenous or endogenous genotoxic molecules including reactive oxygen species (ROS). The other mechanism underlying polyphenol effects consists in inhibition of activity and synthesis of carcinogen-metabolizing enzymes. Plant polyphenols can induce expression of genes encoding antioxidant and detoxification enzymes and this also prevents initiation of carcinogenesis.
Keywords: plant polyphenols; carcinogenesis initiation; inactivation of genotoxic molecules; inhibition of metabolic activation of carcinogens; induction of antioxidant and detoxification enzymes

Specificity of molecular recognition in oligomerization of bacterial L-asparaginases by Yu. V. Mezentsev; A. A. Molnar; N. N. Sokolov; V. B. Lisitsina; M. A. Shatskaya; A. S. Ivanov; A. I. Archakov (124-134).
Bacterial L-asparaginases, which are widely used in the antitumor therapy, act only as homotetramers, because their active sites are located at the interface between the subunits of these enzymes. High stability of aspaginase tetramers is determined by ion pair formation between subunits, and this suggests high specificity of molecular recognition during oligomerization of bacterial L-asparaginases. In this study we have investigated specificity of molecular recognition in oligomerization of some bacterial asparaginases by a biosensor based on surface plasmon resonance. It was shown, that a stable tetrameric complex could be formed only by the subunits of the same L-asparaginase. Using two mutants of Helicobacter pylori L-asparaginase it was shown that even a single point mutation at the interface of highly homologous and closely related subunits significantly reduced specificity of molecular recognition.
Keywords: specificity; molecular recognition; oligomerization; L-asparaginase; computer modeling; optical biosensor; surface plasmon resonance

Proteins-markers of placental insufficiency by T. N. Pogorelova; V. O. Gunko; N. A. Drukker; V. A. Linde (135-138).
The proteomic analysis of amniotic fluids of women with physiological pregnancy and pregnancy, complicated by placental insufficiency has been carried out on the second and the third trimesters. The differences in protein patterns between physiological pregnancy and pregnancy complicated by placental insufficiency seen during these gestation periods include: i) the absence of peroxiredoxin 2, epidermal fatty acid binding protein, and haptoglobin in placental insufficiency; ii) appearance of several proteins absent in physiological pregnancy: hippocalcin-like protein 1, CDC37-like protein, NKG2D ligand 2 (II trimester), CDC37-like protein, NKG2D ligand 2 (III trimester). These differences in the amniotic fluid proteome, obviously, have pathogenetic importance for the development of the placental insufficiency. The revealed proteins of distinction may serve as markers of this obstetrical pathology.
Keywords: proteomic analysis; different protein patterns; placental insufficiency; amniotic fluids

Comparative thermodynamic analysis of thrombin interaction with anti-thrombin aptamers and their heterodimeric construct by S. Yu. Rakhmetova; S. P. Radko; O. V. Gnedenko; N. V. Bodoev; A. S. Ivanov; A. I. Archakov (139-143).
Aptamers interacting selectively with the anion-binding exosites 1 and 2 of thrombin were merged into dimeric oligonucleotide constructs by means of a poly-(dT)-linker of 35 nucleotides (nt) in length. Complexes of thrombin with the aptamers and their hetero- and homodimeric constructs were measured using an optical biosensor Biacore-3000. The K D values obtained for the hetero- and homodimeric constructs were correspondingly 25–30- and 2–3-fold lower than those for the primary aptamers. Analysis of temperature dependencies of the K D values within the temperature interval of 10–40°C has shown that affinity increases with the temperature decrease. The values of the enthalpy change ΔH upon formation of complexes of thrombin with the aptamers and the heterodimeric construct were basically the same. The value of the entropy change ΔS upon complex formation of thrombin with the aptamer heterodimeric construct was 1.5–2-fold higher than the ΔS values for the complexes with the aptamers. The complex formation and dissociation rates increased with the elevation of temperature from 10 to 37°C. However, at both temperatures the dissociation rate for the complex of thrombin with the heterodimeric construct was evidently lower that for the complexes with the aptamers.
Keywords: aptamers; thrombin; heterodimeric construct; affinity; thermodynamics

Characterization of oligonucleotides with LNA-monomers for PCR detection of point mutations in Mycobacterium tuberculosis genome by O. Yu. Limanskaya; T. N. Fesenko; V. A. Pokrovskiy; T. N. Mukhina; V. N. Stepanshina; I. G. Shemyakin; A. P. Limanskii (144-151).
Point mutations associated with isoniazid resistance in Mycobacterium tuberculosis (MTB) have been analyzed in codon 315 of the katG gene by conventional polymerase chain reaction (PCR) using primers containing locked nucleic acid (LNA) modified nucleotides. Purity and structure of primers containing 5 LNA monomers of 17 nucleotides in length were characterized by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and a 17-mer duplex formed by two complementary oligonucleotides was characterized by the method of thermal denaturation. The duplex containing five LNA monomers per each strand was characterized by a higher melting temperature than it was expected using extrapolation of theoretical calculation for nucleotide modification of one strand of the duplex. Detection of any of six possible mutations in katG codon 315 (i.e. discrimination between sensitive and resistant MTB) requires just one PCR employing a set of two primers with one LNA-modified primer; this is an important advantage of oligonucleotides containing LNA over unmodified nucleotides: employment of multiplex PCR would require up to 12 primers. Problems of control of oligonucleotide modification by LNA monomers are discussed.
Keywords: DNA melting; point mutation; polymerase chain reaction; mycobacteria tuberculosis; locked nucleic acid; mass spectrometry with matrix-assisted laser desorption/ionization

A total mixture of phospho- and glycolipids from sea macrophytes Sargassum pallidum, Ulva fenestrata, Zostera marinawas separated and the fatty acid composition was determined. Biological activity of the mixtures of polar lipids and natural antioxidants (echinochrome A from the flat sea urchin Scaphechinus mirabilis and a polyphenolic complex from the sea grass Zostera marina) were studied under conditions of impairments of carbohydrate and lipid metabolism. Doses and compositions of mixtures of polar lipids and antioxidants possessing high corrective activity were optimized in mice with the experimental model of atherosclerosis and diabetes. Based on these results possible mechanisms of the effects of polar lipids containing various polyunsaturated fatty acids and the investigated antioxidants have been proposed. The developed compositions may be used for creation of new biologically active additives and drugs.
Keywords: polar lipids; luteolin; rosmarinic acid; diabetes mellitus; hyperlipidemia; 1,4-naphtoquinones

Manganese in atherogenesis: Detection, origin, and a role by A. P. Lozhkin; T. B. Biktagirov; V. A. Abdul’yanov; O. V. Gorshkov; E. V. Timonina; G. V. Mamin; S. B. Orlinskii; N. I. Silkin; V. M. Chernov; R. N. Khairullin; M. Kh. Salakhov; O. N. Ilinskaya (158-162).
The role of transition metal ions in atherogenesis is controversial; they may be involved in hydroxyl radical generation and can also catalyze the reactive oxygen species neutralization reaction as cofactors of antioxidant enzymes. Using EPR spectroscopy, we revealed that 70% of aorta specimens with atherosclerotic lesions possessed superoxide dismutase activity, 100% of the specimens initiated Fenton reaction and demonstrated the presence of manganese paramagnetic centers. The sodA gene encoding manganese-dependent bacterial superoxide dismutase was not found in the samples of atherosclerotic plaques by PCR using degenerate primers. The data obtained indicate prospects of manganese analysis as a marker element in the express diagnostics of atherosclerosis.
Keywords: atherosclerosis; superoxide dismutase; manganese; sodA gene

The antioxidant and antiaggregant effects of the covalent bienzyme superoxide dismutase — Chondroitin sulfate — Catalase conjugate on platelets by A. V. Vavaev; L. I. Bouryachkovskaya; E. G. Tischenko; I. A. Uchitel; A. V. Maksimenko (163-170).
A covalent bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate (SOD-CHS-CAT) demonstrated the dose-dependent inhibitory action on induced platelet aggregation both in the presence and the absence of hydrogen peroxide. The antioxidant activity of SOD-CHS-CAT was noted at much lower doses than that of other CAT derivatives. The conjugate SOD-CHS-CAT inhibited platelet aggregation regardless of the nature of inducers; it also prevented platelet spreading on the glass surface. The latter suggests conjugate influence on adhesive properties of platelets. Novel properties of the SOD-CHS-CAT conjugate underlie prospects of its biopharmaceutical applicability as a tool for antioxidant therapy.
Keywords: platelets; aggregation; ADP; hydrogen peroxide; catalase; bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate

Assessment of caspase 3 activity in rabbit myocardial tissue during experimental hemodynamic overload of the left ventricle of the heart by M. L. Blagonravov; M. V. Onufriev; E. A. Demurov; N. V. Gulyaeva; V. A. Frolov (171-174).
It is well known that chronic overload of the cardiac left ventricle is accompanied by an increase in the cardiomyocyte apoptosis rate. However, direction and extent of changes in programmed cell death under an acute overload of the left ventricle still requires detailed investigation (as its pathogenesis significantly differs from chronic overload). Caspase-3 activity has been investigated in left ventricle myocardium of rabbits on days 1, 3, and 5 after modeling of left ventricle hemodynamic overload caused by experimental stenosis of the ascending aorta. Control group included intact animals. It was found that caspase-3 activity significantly increased in both ventricles on day 1; it increased more than twofold above control values on day 3 and decreased up to nearly control values on day 5. Based on these data it was concluded that the acute hemodynamic overload of the left ventricle may be a cause of increased apoptosis in the myocardial tissue of both cardiac ventricles during first days of the pathological process.
Keywords: myocardium; left ventricle; right ventricle; overload; caspase; apoptosis

The content of neurotransmitters and their metabolites was investigated in brain cortex hemispheres, thalamus and brainstem of rats subjected to chronic morphine intoxication (7–21 days). Morphine administration for 7–14 days was accompanied by changes of the catecholamine system functioning, which was the most pronounced in the thalamus and the brainstem. These changes included increased secretion of dopamine and noradrenaline, their decrease in the brain tissue, and an increased content of their metabolites. The changes in serotonin and GABA content were less pronounced and included a decrease of serotonin level and the increase of the GABA content in different periods of opiate administration.
Keywords: morphine; brain; dopamine; noradrenaline; serotonin; GABA

Respiratory and phosphorylation functions of rat brain mitochondria were studied by the polarographic method under conditions of insulin shock in animals and after its treatment with glucose or glutamate (in combination with inhalation of air enriched with 7% CO2 as a hypercapnic gas mixture). Certain differences in the effects of the applied agents were found. Phosphorylation ability of mitochondria did not reach the normal level even one day after both ways of the convulsive state treatment. Some respiratory parameters suggest that unfavorable changes in the respiratory chain functioning mainly occur at the level of the respiratory chain complex I.
Keywords: hypoglycemia; brain; oxidative phosphorylation

A sensitive chemiluminescence method has been proposed for Phenobarbital (PB) determination. It is based on the enhancive effect of PB on the chemiluminescence reaction between luminol and dissolved oxygen in a flow injection system. The chemiluminescence intensity linearly responded to the PB concentrations ranged from 0.05 to 10 ng/ml with the detection limit of 0.02 ng/ml (3σ). At a flow rate of 2.0 ml/ min, the whole procedure of PB determination (including sampling and washing) takes just 0.5 minute, offering the sampling efficiency of 120 per 1 h. The method was applied successfully for the PB assay in pharmaceutical preparations, human urine and serum without any pretreatment with recovery from 95.7 to 106.7% and RSDs of less than 3.0%.
Keywords: Phenobarbital; chemiluminescence; luminel

The study of telomerase activity in gastric cancer by L. V. Svinareva; A. I. Glukhov; O. V. Zimnik; I. I. Bykov; T. V. Khorobrykh; V. I. Shvets (188-192).
Telomerase activity was examined in tissue specimens from patients with gastric adenocarcinomas and gastric lymphoma using a modified TRAP (telomeric repeat amplification protocol) assay. Telomerase activity was found in 16 of 18 (89%) patients with gastric adenocarcinomas and gastric lymphoma, whereas it was not detected in a patient with noncancerous gastric mucosa. Almost all analyzed specimens had “high” and “very high” levels of telomerase activity. Telomerase is almost certainly associated with the process of malignant transformation and it can be an important marker for diagnostics of gastric cancer.
Keywords: gastric adenocarcinoma; carcinogenesis; telomerase; telomerase activity (TA)

The study of activity of blood antioxidant enzymes in HIV infection by V. V. Kostyushov; I. I. Bokal; S. A. Petrov (193-196).
The HIV infection is accompanied by activation of lipid peroxidation, oxidative modification of lipoprotein complexes, and a decrease in activity of blood serum antioxidant enzymes (superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx)). A significant increase of glutathione reductase observed under these conditions is considered as the antioxidant defense function. These changes are observed at already stages of this disease (in HIV carriers), however, they were much more pronounced in patients with manifested forms of AIDS.
Keywords: pro- and antioxidants; oxidative stress; HIV; AIDS

The role of induction and inhibition of nitric oxide synthesis in regulation of blood neutrophils cell death during oxidative imbalance by N. V. Ryazantseva; T. V. Zhavoronok; E. A. Stepovaya; Yu. V. Starikov; V. A. Bychkov (197-202).
Modeling of oxidative stress in vitro with 5 mM H2O2 has demonstrated a protective role of nitric oxide in realization of constitutional blood neutrophil cell death. The NO synthase inductor, L-arginine, and the inhibitor of nitric oxide synthesis, L-NAME, influenced the amount of annexin-positive cells, the content of Bax protein, reactive oxygen species, cyclic nucleotides, and calcium homeostasis in neutrophils under conditions realizing programmed death during oxidative stress in vitro and under acute inflammation. During oxidative stress L-arginine normalized an increased intracellular Ca2+ level and the cAMP/cGMP ratio by increasing the cGÌP level, stabilized metabolism and prolonged neutrophil lifetime. During acute inflammation NO induction was insufficient for limitation of Ca2+ release into cytosol and for onset of the apoptotic effect; blockade of NO synthesis deteriorated this situation by activating neutrophil apoptosis due to the sharp increase in the Ca2+ content and reduction of cytosolic cyclic nucleotides. The protective effect of NO on neutrophil cell death during oxidative imbalance was not associated with regulation of the proapoptotic protein Bax.
Keywords: nitric oxide; apoptosis; neutrophils; reactive oxygen species; oxidative stress; cyclic nucleotides