Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.5, #3)

Variability of the healthy human proteome by N. A. Pakharukova; L. Kh. Pastushkova; S. A. Moshkovskii; I. M. Larina (203-212).
The aim of this review is to analyze results of studies on characteristics of protein variability and diversity of posttranslational modifications of proteins in healthy humans. Numerous studies have demonstrated that a proteomic profile is characterized by significant intra- and inter-individual variability, and quite often natural (“normal”) variability of some proteins can be comparable to changes observed in pathological processes. Results obtained by our research group have demonstrated high intra-individual variability of serum low-molecular subproteome of healthy volunteers, certified by a special medial committee (the coefficient of variation (CV) of 42.6%). The proteins characterized by high variability under normal conditions (e.g. haptoglobin — 0–40 mg/ml; lysozyme — 0.01–0.1 mg/ml; C-reactive protein — 0.01–0.3 mg/ml) cannot be considered as potential biomarkers of diseases. On the contrary, proteins and peptides characterized by insignificant dispersion in healthy population (such as albumin ( CV = 9%); transferrin-(CV = 14%); C3c complement (CV = 17%), α-1 acid glycoprotein (CV = 21%), α-2-macroglobulin (CV = 20%); transthyretin fragment (CV = 28.3%) and α2-HS-glycoprotein βchain (CV = 29.7%)) can provide valuable information about the state of health. Thus, studies of plasticity in the proteomic profiles of healthy humans will help to correct reference intervals used in clinical proteomics.
Keywords: variability; proteomic profile; healthy humans

Linear and nonlinear QSAR models of acute intravenous toxicity of organic chemicals for mice by O. A. Raevsky; E. A. Liplavskaya; A. V. Yarkov; O. E. Raevskaya; A. P. Worth (213-225).
The QSAR analysis of acute intravenous toxicity for mice of 68 monofunctional chemical compounds is presented. These compounds are referred to seven chemical classes: hydrocarbons (6 chemicals), alcohols (13), amides (22), amines (12), ethers (5), ketones (7), and nitriles (3). Preliminary consideration of data for these chemical compounds showed the necessity of consideration of nonlinear toxicity — descriptor relationships in addition to linear toxicity — descriptor relationships. The linear and nonlinear QSAR models were considered for each indicated class of organic chemical compounds. Analogical models were constructed for the whole set of the monofunctional chemical compounds. The statistical parameters and robustness of nonlinear models were better than those for linear models. Replacement of a lipophilicity parameter for descriptors characterizing molecular weight and H-bond ability in nonlinear models also improved their statistical characteristics. It was concluded that in the case of nonlinear dependence between the intravenous toxicity of monofunctional chemical compounds and their lipophilicity one may expect nonlinear behavior of this dependence for compounds containing more than one functional group. This conclusion was further checked by generating linear, parabolic, and bilinear models for small clusters containing structurally related compounds with several functional groups and several clusters where nonlinear QSAR models exhibited better statistical criteria compared with linear models were found.
Keywords: linear and nonlinear QSAR models; HYBOT; acute toxicity for mice

We have investigated the effect of polycyclic aromatic hydrocarbons (PAHs) on expression of the estrogen-metabolizing genes CYP1A1, CYP1B1, CYP19 and also ERα, and cyclinD1 genes, regulating cell division in estrogen-depended tissues. Treatment of rats with benzo(a)pyrene (BP) or 3-methylcholantrene (MCA) significantly up-regulated CYP1A1, CYP1B1 gene expression in liver, uterus and ovary, whereas α-naphthoflavone (α-NF) did not have any effect. The high level of aromatase gene (CYP19) expression was detected in ovary only. Treatment of rats with BP or MCA significantly down-regulated expression of this gene (15- and 5,5-fold, respectively), whereas α-NF was ineffective. Administration of BP but not MCA or α-NF increased ERα and cyclinD1 gene expression in rat liver. The levels of ERα and cyclinD1 mRNA levels remained unchanged in uterus of after treatment of rats with these PAHs. BP administration increased ERα and cyclinD1 mRNA levels (3,5- and 2,5-fold, respectively) in ovary, while MCA and α-NF were ineffective. Thus, our results give evidence for tissue-specific effects of PAHs on expression of genes, which participate in hormonal carcinogenesis. On the other hand, the fact that BP and MCA treatments influenced the expression of estrogen-metabolizing genes and genes, which control cell division, supports the viewpoint that PAHs may be one of the causes of endocrine disorders and subsequent hormonal carcinogenesis.
Keywords: CYP1A1; CYP1B1; CYP19; ERα; cyclinD1; hormonal carcinogenesis

Mechanisms of suppression of carcinogenesis promotion/progression by plant polyphenols have been considered. They can exhibit antiproliferative activity by decreasing cyclins and cyclin dependent kinases and by activating their inhibitor proteins in tumor cells. Plant polyphenols can also induce apoptosis by modulating activity of proteins involved in apoptosis and thus exhibit proapoptotic effect. They may inhibit tumor metastasis and angiogenesis. Polyphenols act through the regulation of cell signal transduction and gene expression and exhibit either up or down regulation of genes controlling tumor development.
Keywords: plant polyphenols; carcinogenesis promotion/progression; cell cycle arrest; apoptosis induction; tumor metastasis and angiogenesis; modulation of cell signal transduction

Involvement of integrin α5β1 in apoptosis and drug resistance of human breast carcinoma cells by G. E. Morozevich; N. I. Kozlova; N. A. Ushakova; M. E. Preobrazhenskaya; A. E. Berman (241-245).
The doxorubicin-resistant MCF-7Dox cell line derived from the drug-sensitive MCF-7 human breast carcinoma cell line, differs from the latter by a strongly reduced expression of the α2β1 integrin and a highly increased ex-pression of the α5β1 integrin. Silencing of this integrin in the MCF-7Dox cells by transfection with α5-specific siRNA markedly stimulated anoikis and increased sensitivity of these cells to doxorubicin. α5β1 silencing was also accompanied by a significant inhibition of the activity of signal protein kinases Akt and Erk2. Our results suggest involvement of common integrin-mediated signal mechanisms controlling apoptotic cell death under various stress conditions.
Keywords: integrins; anoikis; tumor growth; drug resistance; signal protein kinases

In most serpentine type receptors the third intracellular loop (ICL-3) is responsible for the interaction with heterotrimeric G proteins and for transduction of a hormonal signal to the enzymes, generators of the second messengers. It was found that the peptides corresponding to ICL-3 influence functional activity of hormonal signaling systems in the absence of the hormone and, consequently, may be considered as prototypes for the development of selective regulators of these systems. We have originally synthesized peptides corresponding to the C-terminal regions 255–269 and 240–254 of ICL-3 of type 1 and 2 rat somatostatin receptors (Som1R and Som2R). Micromolar concentrations of these peptides activated G i proteins and inhibited forskolin-stimulated activity of adenylyl cyclase (AC) in rat brain tissues. The peptide 255–269 of Som1R is a selective antagonist of Som1R, and the peptide 240–254 of Som2R is an agonist of Som1R. The peptide 255–269 of Som1R decreased the regulatory effects of somatostatin and the selective Som1R agonist, CH-275, realized via the homologous receptor, while the peptide 240–254 of Som2R, on the contrary, increased the AC inhibitory effect of CH-275. Both peptides insignificantly influenced regulatory effects of the Som2R agonist octreotide. Thus, the peptides studied by us are selective regulators of the somatostatin-sensitive AC system. Using the peptides we have demonstrated that ICL-3 of both Som1R and Som2R includes the main molecular determinants that are responsible for activation of G i proteins and regulation of the AC system by somatostatin and its analogues.
Keywords: adenylyl cyclase; peptide; somatostatin; somatostatin receptor; third intracellular loop; G-protein

The reaction of adrenaline autooxidation in an alkaline carbonate buffer followed by formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinone pathway of adrenaline metabolism in the body, is accompanied by oxygen consumption. A study of this process by the polarographic method enabled one to apply this reaction to determine the activity of superoxide dismutase and antioxidant properties of biological and chemical compounds; it is based on evaluation of a latent period and the rate of oxygen consumption, which are measured in the presence of examined compounds. It was suggested that known neuroand cardiotoxicity of the quinone products of adrenaline oxidation may be associated not only to their intrinsic properties and reactive oxygen species formed but also local hypoxia of those regions of the cell and tissue where the quinone oxidation of adrenaline occurs.
Keywords: adrenaline; adrenochrome; superoxide; oxygen; polarography; superoxide dismutase

Synthesis and hypoglycemic activity of Bis(L-malato)oxovanadium(IV) by V. K. Gorodetskii; A. I. Tochilkin; N. F. Belayeva; I. R. Kovelman; B. F. Korovkin (260-262).
In order to create new oral vanadyl organic complexes-based drugs for the treatment of diabetes mellitus we have originally synthesized a biligand vanadyl derivative of L-malic acid (bis(L-malato)oxovanadium(IV)) and investigated its potential as a novel hypoglycemic agent using the experimental streptozotocin-induced diabetes in rats. The oral administration of bis(L-malato)oxovanadium(IV) with drinking water significantly reduced blood and urinary glucose concentrations and also the level of glycated proteins in the streptozotocin-diabetic rats.
Keywords: bis(L-malato)oxovanadium(IV); oxovanadium(IV) complexes; L-malic acid; hypoglycemic activity

Inhibition of NO-dependent soluble human platelet guanylate cyclase by isatin by I. S. Severina; A. Yu. Schegolev; G. V. Ponomarev; A. E. Medvedev (263-267).
Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. Physiologically relevant concentrations of isatin (ranged from 1 nM to 10 μM) did not influence basal activity of soluble human platelet guanylate cyclase (sGC), but caused a bell-shaped inhibition of the NO-activated enzyme. Inhibition of the NO-dependent activation by isatin did not depend on a chemical nature of the NO donors. The inhibitory effects of ODC (a heme-dependent inhibitor of sGC) and isatin were non-additive suggesting that the inhibitory effect of isatin may involve the heme binding domain (possibly heme iron) and experiments with hemin revealed some isatin-dependent changes in its spectrum. Isatin also inhibited sGC activation by the allosteric activator YC-1. It is suggested that the bell shaped inhibition of the NO-dependent activation of sGC by isatin may be attributed to complex interaction of isatin with the heme binding domain and the allosteric YC-1-binding site of sGC.
Keywords: guanylate cyclase; nitric oxide (NO); isatin

Anticoagulant and anticomplement effects of an endogenous inhibitor from hepatopancreas of red king crab (Paralithosed camtschaticus) on human blood by M. V. Balashova; L. V. Lyutova; Yu. A. Rudenskaya; V. A. Isayev; S. S. Andina; L. V. Kozlov; G. N. Rudenskaya (268-275).
Serpins are the superfamily of serine and cysteine protease inhibitors (SERine Protease Inhibitors) acting by an irreversible suicide mechanism. A novel serpin from hepatopancreas of red king crab (Paralithosed camtschaticus) was isolated and its effect on the process of human blood plasma coagulation was investigated. The investigated serpin exhibited a significant anticoagulant effect, which dramatically increased in the combination with heparin. The study of the crab serpin on C1s (C1 esterase) revealed its competition with the C1 inhibitor from blood plasma. Although the inhibitor weakly influenced thrombin activity, inhibition constant for C1s was (2.02 ± 0.71) 10−7 M. Unlike the C1 inhibitor the novel red king crab serpin does not inhibit fibrinolysis but inhibits blood coagulation. This creates certain clinical perspectives.
Keywords: inhibitor from red king crab; serpin; heparin; tromboelastography; anticoagulant activity; the complement system

Liposome formulations of combretastatin A4 and its 4-arylcoumarin analogue prodrugs: The antitumor effect in the mouse model of breast cancer by E. V. Moiseeva; N. R. Kuznetsova; E. V. Svirshchevskaya; N. V. Bovin; N. S. Sitnikov; A. S. Shavyrin; I. P. Beletskaya; S. Combes; A. Yu. Fedorov; E. L. Vodovozova (276-283).
The antimitotic agent combretastatin A-4 (CA-4) has been recently proposed as an antivascular agent for anticancer therapy. In order to reduce systemic toxicity by means of administration in liposome formulations, new lipophilic prodrugs, oleic derivatives of CA-4 and its 4-arylcoumarin analogue (CA4-Ole and ArC-Ole, respectively), have been synthesized in this study. Liposomes with mean diameter of 100 nm prepared on the basis of egg phosphatidylcholine and baker’s yeast phosphatidylinositol quantitatively included up to 15 mol% of CA4-Ole, or 7 mol% of ArC-Ole. To achieve targeting to neovascular endothelium prodrug bearing liposomes decorated with the tetrasaccharide selectin ligand Sialyl Lewis X (SiaLeX) have been also prepared. The antitumor activity was studied in vivo using the model of slow-growing mouse breast cancer. Under the dose used (22 mg/kg) and the administration protocol (four injections, one per a week, starting from the appearance of palpable tumors) cytostatic CA-4 did not reveal any anticancer effect; moreover, it even stimulated tumor growth. The liposome formulations of CA4-Ole did not demonstrate such stimulation. However, to achieve a pronounced antitumor effect, the number of injections of liposomes should be apparently increased. The cytotoxic activity of a novel antimitotic agent ArC was one order of magnitude lower in the human breast carcinoma cell culture in vitro. Nevertheless, in vivo in the mouse model of breast cancer the antitumor effect of this compound corresponded to the double equivalent dose of CA-4. The results demonstrate perspectives of SiaLeX-liposomes loaded with ArC-Ole: the preparation partially inhibited tumor growth already after the second injection. Thus, subsequent optimization of doses and regimens of administration both for ArC and liposomal ArC-Ole formulations are needed.
Keywords: combretastatin A-4; 4-arylcoumarins; lipophilic prodrugs; liposomes; Sialyl Lewis X; breast cancer

The influence of glucose on free radical peroxidation of low density lipoproteins in vitro and in vivo by V. Z. Lankin; G. G. Konovalova; A. K. Tikhaze; L. V. Nedosugova (284-292).
In the range of concentrations 12.5–100 mM glucose stimulated Cu-mediated free radical peroxidation of low density lipoproteins (LDL) from human blood plasma. Based on analysis of kinetic parameters of the LDL peroxidation it was found that intensification of this process is caused by formation of free radical intermediates of glucose autooxidation during generation of reactive oxygen species in the presence of transition metal ions. Normalization of blood glucose in patients with type 2 diabetes during therapy was accompanied by a significant decrease of LDL oxidation. Therapy with the sugar-lowering drug metformin, which utilizes methylglyoxal, caused much higher inhibition of the in vivo LDL peroxidation in blood of patients with diabetes mellitus probably due to the decrease of methylglyoxal-dependent generation of superoxide anion radicals shown by us earlier [Biochemistry (Moscow) 2009, vol. 74, pp. 568–574].
Keywords: oxidative stress; carbonyl stress; modified low density lipoproteins; glucose; lipohydroperoxides; malondialdehyde; methylglyoxal; metformin

Interrelationship between total, multimer (MM) and oligomer (OM) adiponectin blood concentrations with some metabolic parameters has been investigated in 49 men and 49 women (mean age of 57.3 ± 10.1 years). We have found negative correlations between total blood adiponectin and its MM form content with body mass index, waist circumference, the insulin resistance index HOMA, with blood concentrations of insulin, glucose, free fatty acids, triglycerides and also positive correlations with high density lipoprotein cholesterol content. There was a poor correlation between OM adiponectin concentration with any parameter studied. According to the regression analysis, concentration of total adiponectin but not its MM form was an independent determinant of the HOMA index in women and free fatty acid concentration in men. In the group of men with the low level of adiponectin its MM form but not total adiponectin reversely correlated with the HOMA index and was its independent determinant. Thus, correlation between blood adiponectin concentration and metabolic parameters is associated with its MM rather than OM form. Study of the role of adiponectin in development of metabolic disorders may be limited to determination of total blood adiponectin concentration except a group of male patients characterized by a low level of this adipokine. In these patients concentrations of the MM form should be determined.
Keywords: adiponectin; insulin resistance; metabolic syndrome

Peculiarities of functional activity of circulating phagocytes in patients with different forms of drug-resistant pulmonary tuberculosis by O. T. Titarenko; M. E. Dyakova; D. S. Esmedlyaeva; M. V. Pavlova; A. V. Yelkin; N. P. Alekseeva; B. B. Bondarenko (301-306).
Functional activity of circulating phagocytes (macrophages and neutrophils — Ns) was studied in 30 patients with infiltrative and 30 patients with fibro-cavernous pulmonary tuberculosis (IPT and FCPT, respectively), characterized by similar biological properties of mycobacterium tuberculosis (MTB), dissemination of the process, and manifestations of intoxication. Differences of the functional activity of both types of cells depending on the PT form were found: a more significant increase in the oxygen-depending activity in FCPT while bactericide potential estimated with the zymosan induced NST-test was more pronounced in IPT patients. These data correlate with the blood levels of neopterin and elastase, the markers of the mononuclear and neutrophil activity, respectively. Involvement of adenosine deaminase (ADA) and neopterin in realization of intracellular oxygen-dependent processes was demonstrated. Results of the multivariate analysis of the whole set of the studied phagocyte parameters, reflecting their different roles in this pathological process demonstrated a prevailing role of mononuclears in newly diagnosed IPT and neutrophils in the chronic progressive process.
Keywords: tuberculosis; drug-resistance; phagocyte activity; NST-test; adenosine deaminase; neopterin

Increased myeloperoxidase activity is a risk factor for ischemic heart disease in patients with diabetes mellitus by I. V. Gorudko; V. A. Kostevich; A. V. Sokolov; I. V. Buko; E. E. Konstantinova; N. L. Tsapaeva; E. V. Mironova; E. T. Zakharova; V. B. Vasilyev; S. N. Cherenkevich; O. M. Panasenko (307-312).
Using a previously developed spectrophotometric method (Bioorg. Khim. 2009, vol. 35, pp. 629–639) a significant increase of myeloperoxidase (MPO) activity (versus healthy control) was found in blood plasma of patients with type 2 diabetes mellitus (DM2) without cardiovascular complications, and also in patients with ischemic heart disease (IHD). The plasma MPO concentration measured by an enzyme-linked immunosorbent assay was significantly higher only in blood plasma of patients with DM2 and IHD. A significant positive correlation between blood MPO activity and blood MPO content was observed only in blood plasma samples from healthy donors. Increased MPO activity did not correlate with MPO concentration in blood plasma of patients with DM2 and DM2 with IHD. Taken together, these results indicate that studies on the MPO role in the development of pathological processes should include simultaneous determination of both the amount of enzyme and its peroxidase activity in blood of patients. The proposed approach gives comprehensive information about the relationship between MPO activity and MPO concentration in patient’s blood. Since the high concentration of MPO is a diagnostically important parameter for the prediction of development of endothelial dysfunction and cardiovascular diseases, the obtained results point to the contribution of MPO-dependent reactions in cardiovascular complications associated with diabetes mellitus. MPO activity may serve as an additional diagnostic criterion for determination of risk of IHD in DM patients.
Keywords: myeloperoxidase; diabetes mellitus; neutrophils; peroxidase activity; blood plasma; enzyme-linked immunosorbent assay