Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.6, #2)

Protocols of protein interactomics: Molecular fishing on optical chips and magnetic nanoparticles by A. S. Ivanov; P. V. Ershov; Yu. V. Mezentsev; E. V. Poverennaya; A. V. Lisitsa; A. I. Archakov (99-106).
In the living systems proteins function through interactions, which produce stable and dynamic protein complexes. Therefore necessity of profound studies of protein functions stipulates expansion of protein-protein interaction research. In the present review we describe experimental methods and protocols of protein interactomics, based on technology of molecular fishing on optical chips and paramagnetic nanoparticles. Both approaches are comparatively evaluated in the present review.
Keywords: interactomics; protein-protein interaction; proteomics; molecular fishing; optical biosensor; magnetic particles; protein mass-spectrometry

Conotoxins: From the biodiversity of gastropods to new drugs by A. E. Fedosov; S. A. Moshkovskii; K. G. Kuznetsova; B. M. Olivera (107-122).
The review describes general trends in research of conotoxins, the peptide toxins isolated from sea gastropods of the genus Conus. It covers publications from conotoxin discovery in 1970th up to contemporary research and considers classification of conotoxins, their structural diversity and different ways of their action on molecular targets, particularly, ion channels. Special attention is paid to the applied aspect of conotoxin research especially to perspectives of the development of conotoxin based drugs. The first example of such conotoxin based drug is ziconotide an analgesic of a new generation.
Keywords: conotoxin; conopeptide; gastropod; ion channel; drug substance

Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect by M. V. Pokrovskaya; V. S. Pokrovskiy; S. S. Aleksandrova; N. Yu. Anisimova; R. M. Andrianov; E. M. Treschalina; G. V. Ponomarev; N. N. Sokolov (123-131).
The recombinant producer strain expressing Rhodospirillum rubrum L-asparaginase (RrA) has been obtained and a purification procedure of RrA has been developed. The purified enzyme, RrA, has the following biochemical and catalytic characteristics: Km for L-Asn of 0.22 mM, pH optimum at 9.2; temperature optimum at 54°C, pI = 5.1. RrA exhibited a significant cytotoxic effect towards the following cell lines: K562 (IC50 = 1.80 U/mL), DU145 (IC50 = 9.19 U/mL), and MDA-MB-231 (IC50 = 34.62 U/mL). Comparative analysis employing E. coli L-asparaginase II type (EcA) and Erwinia carotovora L-asparaginase (EwA) has shown that the enzyme cytotoxicity towards these cell lines decreased in the following order: EcA > RrA > EwA. Daily administration of RrA (4000 U/kg) to L5178y bearing mice for 10 days (total dose of 40000 U/kg) showed T/C = 172. Data obtained suggest that RrA may be referred to intracellular L-asparaginases with low L-glutaminase activity and marked antiproliferative effect.
Keywords: Rhodospirillum rubrum ; intracellular L-asparaginase; antiproliferative activity

Electrochemical sensor systems based on one-dimensional (1D) nanostructures for analysis of bioaffinity interactions by V. V. Shumyantseva; T. V. Bulko; E. V. Suprun; A. I. Archakov (132-137).
Modification of screen printed graphite electrodes (SPE) with gold nanoparticles (AuNPs) decorated Pb nanowires (PbNWs) enhances such analytical characteristics of the sensor as effective surface area, electrocatalytic properties and kinetics of heterogeneous electron transfer. The reason for such improvement may be associated with the synergistic effect of AuNPs and PbNWs. Nanowires ensembles on the electrode surface were employed for the detection of cardiac myoglobin in human blood, cytochrome P450 2B4, cytochrome c. Composite materials based on nanoparticles with different dimensions (three dimensional (3D) gold nanoparticles and one dimensional (1D) Pb nanowires) create a platform for electrochemical analysis of proteins with low detection limits.
Keywords: Pb nanowires; gold nanoparticles; synergism; nanowires ensembles on electrode

Salivary gland secretions of three species of the medicinal leech differ in the level of their lysozyme, and peptidoglycan-lysing activity. Using a synthetic fluorogenic substrate, 4-methylumbelliferyltetra N-acetyl-β-chitotetraoxide, the glycosidase activity (as one of peptidoglycan-lysing activities) of salivary gland secretion of these species of the medicinal leech was quantitatively evaluated in comparison with egg lysozyme. It is suggested that lysozyme activity of the leech secretions is determined not only by 5 isoforms of destabilase-lysozyme, but by some other enzymes which can utilize these substrates including lysozymes other than i-type (invertebrate) lysozymes.
Keywords: medicinal leech salivary gland secretion; lysozyme activity; glycosidase activity; substrate 4-methylumbelliferyl-tetra N-acetyl-β-chitotetraoxide; egg lysozyme; destabilase-lysozyme

The effect of oxidized dextrans on generation of reactive oxygen species by murine peritoneal exudate phagocytes by V. O. Tkachev; M. V. Zaikovskaya; A. V. Troitsky; N. G. Luzgina; V. A. Shkurupy (144-148).
The effects of oxidized dextrans of different molecular masses on reactive oxygen species production and mitochondrial transmembrane potential of macrophages and neutrophils have been studied in vivo and in vitro. Oxidized dextrans demonstrated moderate direct antioxidant properties but induced intracellular oxidative stress through the increase of oxygen radical generation. This effect of the investigated compounds amplifies the cytotoxic and bactericidal potential of phagocytes and in addition it can influence isoniazid metabolism, thus increasing its efficiency in therapy of infectious diseases.
Keywords: oxidized dextrans; reactive oxygen species; macrophages; neutrophils; mitochondria

Entrapment and in vitro release of delta-sleep inducing peptide from polymer hydrogels based on modified polyvinyl alcohol by T. V. Sukhanova; A. A. Artyukhov; I. A. Prudchenko; A. C. Golunova; M. A. Semenikhin; I. Shtilman; E. A. Markvicheva (149-155).
The aim of this study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to determine kinetics of the peptide release from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels based on poly(vinyl alcohol) acrylic derivative (Acr-PVA) and also macroporous epoxy groups containing hydrogels synthesized by copolymerization of this macromer and glycidyl methacrylate, have been used in this study. Isotropic hydrogels were prepared at positive temperatures while macroporous ones were obtained by formation in cryo-conditions. The peptide was entrapped into macroporous PVA hydrogels by adding the peptide solution onto preformed matrices, while peptide immobilization on PVA-GMA hydrogels, containing free epoxy groups, was carried out by sorption of peptide from its aqueous solution. In the case of DSIP entrapment into isotropic PVA gel the peptide solution was added into the polymer mixture at hydrogel formation. The kinetics of peptide release from hydrogels was studied by incubating matrices in PBS solution (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by reverse-phase HPLC. Incubation of macroporous PVA gels in PBS, 0.9% NaCl, and water for 30 min caused release of 74, 70, and 64% DSIP, respectively, and this processes completed within 3 h. From hydrogel containing epoxy groups the release of neither peptide nor its degradation products was observed even after incubation for 48 h. For freshly prepared isotropic hydrogel the release kinetics was as follows: 27 and 78% DSIP were released within first 30 min and 33 h, relatively. For the lyophilized hydrogel samples the peptide release was 63% after incubation for 30 min, while drying of samples at room temperature for 3 days caused significant peptide loss because of its structure damage.
Keywords: macroporous polymer gels; modified PVA; delta-sleep inducing peptide; peptide entrapment; kinetics of peptide release in vitro; tissue engineering

The effects of some ethanol-metabolizing systems (aldehyde dehydrogenase, catalase, cytochrome P450 2E1) on activation of lipid peroxidation (LPO) processes in the gastrointestinal tract of rats have been studied using inhibitors of these systems. The intensity of LPO processes was evaluated by thiobarbituric acid-reactive substances and chemiluminiscence intensity. It was found, that acetadehyde metabolism plays the major role in the LPO induction in epithelium of the rat gastrointestinal tract.
Keywords: ethanol; gastrointestinal tract; lipid peroxidation

The effect of berberine administration to rats on the functional state of liver after common bile duct ligation by I. V. Zverinsky; N. G. Melnichenko; V. A. Poplavsky; I. P. Sutsko; P. G. Telegin; A. G. Shlyahtun (159-163).
On day 8 after ligation of the common bile duct in rats a significant increase in the serum content of total lipids, cholesterol, bilirubin and ALT, alkaline phosphatase, and gamma-glutamyltransferase was observed. In the hepatic microsomal fraction there was a marked decrease in the content and activity of microsomal monooxygenases. Introperitoneal injections of berberine (10 mg/kg) for 6 days caused a partial normalization of hepatocyte plasma permeability and activity of microsomal flavin-containing monooxygenases. It is suggested that berberine is a substrate and inducer of flavin-containing monooxygenases. The membrane-stabilizing effect of berberine is probably realized at the level of inhibition of the prooxidant status of liver cells.
Keywords: cholestasis; berberine; cytochrome P450; flavin-containing monooxygenases

Anticancer activity of oxovanadium compounds by O. Yu. Abakumova; O. V. Podobed; N. F. Belayeva; A. I. Tochilkin (164-170).
Cytotoxic and antitumor activities of the biligand vanadyl derivative of L-malic acid, (bis-(L-malato)oxovanadium(IV) (VO(mal)2), the inorganic vanadium(IV) compound, vanadyl sulfate (VOSO4), the oxovanadium monocomplex with L-malic acid (VO(mal)), and the vanadyl biscomplex with acetylacetonate (VO(acac)2) were investigated using several tumor cell lines: mouse fibrosarcoma (L929), rat pheochromocytoma (PC12), human liver carcinoma (HepG2), mouse embryonic fibroblasts (NIH/3T3), and also normal human skin fibroblasts. The results showed that VO(mal)2 effectively inhibited growth of cancer cell cultures without any toxic effect on normal human skin fibroblasts. The cytotoxic anticancer effect of vanadium complexes depended on concentration of the compounds studied, incubation time, types of cell cultures, and nature of ligands surrounding the central group of the complex (VO2+). These studies provide evidence that VO(mal)2 may be considered as a potential anticancer agent due to its low toxicity for non-tumor cells and significant anticancer activity.
Keywords: oxovanadium compounds; bis(L-malato)oxovanadium(IV); cell culture; cytotoxicity; anticancer activity

The effect of guanidine derivatives on citrate content and activity of aconitate hydratase and citrate synthase have been investigated in rats with cerebral ischemia-reperfusion. Administration of N-[imino(1-piperidinyl)methyl]guanidine and N-[imino(4-morpholinyl)methyl]guanidine resulted in changes of specific activities of aconitase and citrate synthase towards control values. Under these conditions the citrate level considerably decreased versus rats with untreated ishemia-reperfusion. Administration of these biguanidines compounds also decreased the degree of DNA fragmentation, which was markedly increased in rats with ischemia-reperfusion. The dose-dependent effects of guanidine derivatives suggest that they exhibit not only antioxidant but also prooxidant effects.
Keywords: brain; ischemia-reperfusion; biguanides; rats

The influence of mildronate on peripheral neuropathy and some characteristics of glucose and lipid metabolism in rats with the streptozotocin model of diabetes mellitus by J. Sokolovska; J. Rumaks; N. Karajeva; D. Grinvalde; J. Sharipova; V. Kluša; I. Kalvinsh; N. Sjakste (177-184).
Rats with the streptozotocin (STZ) model of diabetes mellitus were treated with mildronate (100 mg/kg daily, per os or intraperitoneally) for 6 weeks. Body weight, blood glucose, triglycerides, ketone body concentrations, percent of glycated hemoglobin (HbA1c%), glucose tolerance, and the development of neuropathic pain were monitored throughout the whole experiment. The mildronate treatment completely prevented the development of the diabetic neuropathy from the first week up to the end of experiment. In the group of diabetic animals treated with mildronate a significant decrease of blood glucose was observed on the fourth week of the treatment, the level of triglycerides decreased from the third to sixth weeks. Mildronate also decreased accumulation of glycated hemoglobin on the sixth week and improved glucose tolerance compared with untreated animals. The data obtained confirm applicability of mildronate for therapy of diabetes mellitus and its complications.
Keywords: mildronate; streptozotocin; diabetes mellitus; peripheral neuropathy

Using N-acetylglucosamine and N-acetylgalactosamine as model agents for glycation of native hyaluronidase and its chondroitin sulfate modified form it has been shown that the modified enzyme exhibited higher inactivation than the native enzyme, while heparin caused similar inhibition of both forms. Such effect could be attributed to the development of electrostatic interactions as the modified hyaluronidase had altered surface electrostatic potential after chondroitin sulfate binding. However, variations in ionic strength of the medium containing enzyme derivatives have shown that their endoglycosidase activity changed in a similar manner and the effect on glycation represents a multifactor process. N-acetylhexosamines are natural labels of endothelial glycocalyx degradation products. Interaction of the hyaluronidase forms with charged hyaluronan fragments revealed significantly higher inactivation of the modified enzyme compared with the native enzyme. The glycation pattern observed in this study was opposite to that observed with mono- and disaccharides. Thus, it appears that the investigated hyaluronidase derivatives represent an informative enzymatic test in vivo for determination of the dominant type of glycation agents in blood circulation and their origin.
Keywords: glycation; N-acetylhexosamines; hyaluronidase; chondroitin sulfate; electrostatic interactions; ionic strength of medium; hyaluronan fragments

Modification of placental blood serum proteins induced by low temperatures by O. V. Falko; N. G. Zemlianskykh; O. V. Lipina; O. S. Prokopyuk (192-200).
Changes in physical and chemical factors appeared in response to freeze-thawing and low temperature storage of biological samples can result in impairments of protein structures. Spontaneous and diamide-induced protein aggregation of placenta blood serum stored at −20 and −196°C up to 2 years has been investigated by SDS-PAGE. It was shown that storage of placental blood serum at low temperatures did not cause any quantitative and qualitative changes in fraction distribution of proteins denatured by SDS compared with native (unfrozen) samples. Application of β-mercaptoethanol revealed that during freeze-thawing placental blood serum proteins did not form spontaneous aggregates cross-linked by disulphide bridges. Oxidation of amino acid sulfhydryl groups induced by diamide and accompanied by formation of high molecular aggregates was a reasonably effective approach for indirect assessment of structural changes in protein molecules induced by low temperatures. In the samples exposed to low temperature storage protein aggregation induced by 4 mM diamide was significantly higher than in native serum. The structural changes in serum proteins caused by low temperatures and recognized by discrepant susceptibility to diamide-induced protein aggregate formation did not depend on temperature (−20 and −196°C) and time-length of storage (2 years and 3 weeks). These changes do reflect protein reaction to freeze-thawing processes and could originate from ice crystal formation which takes place in unprotected media.
Keywords: proteins; electrophoresis; placenta blood serum; freezing; diamide

The diagnostic value of RNA oncomarkers in evaluation of malignant breast tumors by A. G. Globa; Ya. I. Alekseev; D. A. Varlamov; A. A. Vishnevsky (201-203).
The levels of the RNA oncomarkers, telomerase (hTERT), cytokeratin-19 (CK-19), and mammaglobin (MAM) have been investigated in capillary blood of female patients with mammary ductal carcinoma. The study revealed overexpression of all three factors in patients with this pathology. This overexpression was not found in healthy donors and female patients with mammary fibroadenoma. Levels of the RNA oncomarkers return to the normal level within 10 days after successful tumor resection. These results have been used for the development of diagnostic kits, which may be applicable for differential diagnostics, screening and postoperation monitoring of patients with malignant breast tumors.
Keywords: polymerase chain reaction; telomerase; mammaglobin; oncomarkers; carcinoma