Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.10, #3)

Remodeling of angiogenesis and lymphangiogenesis in cervical cancer development by O. V. Kurmyshkina; L. L. Belova; P. I. Kovchur; T. O. Volkova (191-211).
The ability to stimulate angiogenesis/lymphangiogenesis is an intrinsic property of cancer cells, providing them necessary conditions for growth and metastasis. The “angiogenic switch” is one of the earliest consequences of malignant transformation; it involves altered expression of numerous genes and triggers a complex set of signaling pathways in endothelial cells. Processes of tumor microvascular network formation are closely associated with the stages of carcinogenesis (from appearance of benign lesions to invasive forms) and occur with numerous deviations from the norm. Analysis of expression of proangiogenic factors during sequential steps of cervical cancer development (intraepithelial neoplasia, cancer in situ, microinvasive, and invasive cancer) provides opportunity to reconstruct the regulatory mechanisms of angiogenesis/lymphangiogenesis with emphasis on the most important components. This review summarizes literature data on expression of key regulators of angiogenesis in cervical intraepithelial neoplasia and cervical cancer and analyses their possible involvement in molecular mechanisms of neoplastic transformation of epithelial cells, as well as invasion and tumor metastasis. Correlation between expression of proangiogenic molecular factors and various clinicopathological parameters is considered in the context of their possible use in molecular diagnostics and targeted therapy of cervical cancer. Special attention is paid to rather poorly studied regulators of lymphangiogenesis and “non-VEGF dependent,” or alternative, angiogenic pathways that constitute the prospect of future research in the field.
Keywords: angiogenesis; lymphangiogenesis; cervical cancer; intraepithelial neoplasia; vascular growth factors; transcription factors

Oxidative stress in adipose tissue as a primary link in pathogenesis of insulin resistance by D. I. Kuzmenko; S. N. Udintsev; T. K. Klimentyeva; V. Yu. Serebrov (212-219).
Obesity is a leading risk factor of diabetes mellitus type 2 (DM2), impairments of lipid metabolism and cardiovascular diseases. Dysfunctions of the accumulating weight of the visceral fat are primarily linked to pathogenesis of systemic insulin resistance. The review considers modern viewpoints on biochemical mechanisms underlying formation of oxidative stress in adipocytes at obesity, as one of key elements responsible for impairments of their metabolism triggering formation of systemic insulin resistance.
Keywords: adipocyte; oxidative stress; insulin resistance; diabetes

The program Proteocat as a tool for planning of proteomic experiments by V. S. Skvortsov; N. N. Alekseychuk; D. V. Khudyakov; A. V. Mikurova; A. V. Rybina; S. E. Novikova; O. V. Tikhonova (220-226).
ProteoCat is a computer program that has been designed to help researchers in the planning of large-scale proteomic experiments. The central part of this program is the unit of hydrolysis simulation that supports 4 proteases (trypsin, lysine C, endoproteinases Asp-N and GluC). For peptides obtained after virtual hydrolysis or loaded from data files a number of properties important in mass-spectrometric experiments can be calculated and predicted; the resultant data can be analyzed or filtered (to reduce a set of peptides). The program is using new and improved modifications of own earlier developed methods for pI prediction, which can be also predicted by means of popular pKa scales proposed by other reseachers. The algorithm for prediction of peptide retention time has been realized similarly to the algorithm used in the SSRCalc program. Using ProteoCat it is possible to estimate the coverage of amino acid sequences of analyzed proteins under defined limitation on peptides detection, as well as the possibility of assembly of peptide fragments with user-defined minimal sizes of “sticky” ends. The program has a graphical user interface, written on JAVA and available at http://www.ibmc.msk.ru/LPCIT/ProteoCat.
Keywords: mass spectrometry; peptide properties prediction; protein hydrolysis; protein sequence coverage

The influence of gravity discharge on the content of isatin-binding proteins in mice: The results of ground-based and space research under the program BION-M no. 1 by A. S. Ivanov; A. E. Medvedev; O. A. Buneeva; O. V. Gnedenko; P. V. Ershov; Y. V. Mezentsev; E. O. Yablokov; L. A. Kaluzhskiy; A. V. Florinskaya; N. E. Moskaleva; V. G. Zgoda (227-229).
Using the methods of biosensor and proteomic analysis isatin-binding activity of liver proteins has been investigated in mice from the control and flight groups. Isatin-binding activity was higher in mice of the flight group. The content of certain individual isatin-binding proteins in the samples of the flight groups insignificantly differed from the ground control. However, in samples from animals exposed to weekly postflight adaptation, the level of certain proteins was significantly increased. The latter suggests that the main events in the proteome of mice (at least in subproteome of isatin-binding proteins) occur in the early postflight period.
Keywords: gravitational unloading; BION (satellite); mice; proteome; isatin-binding proteins; biosensors; mass spectrometry

Proteomic analysis of exhaled breath condensate for diagnostics of respiratory system diseases by A. S. Kononikhin; K. Yu. Fedorchenko; A. M. Ryabokon; N. L. Starodubtseva; I. A. Popov; M. G. Zavialova; E. C. Anaev; A. G. Chuchalin; S. D. Varfolomeev; E. N. Nikolaev (230-234).
Study of proteomic composition of exhaled breath condensate (EBC) is a promising non-invasive method for diagnostics of respiratory system diseases in patients. In this study the EBC proteomic composition of 53 donors, including patients with different respiratory system diseases has been investigated. Cytoskeletal keratins type II (1, 2, 3, 4, 5, 6) and cytoskeletal keratins type I (9, 10, 14, 15, 16) were invariant for all samples. Analyzing the frequency of occurrence of proteins in different groups of examined patients, several categories of proteins have been recognized: proteins found in all pathologies (dermcidin, alpha-1- microglobulin, SHROOM3), proteins simultaneously found in two groups (CSTA, LCN1, JUP, PIP, TXN), and proteins specific for a particular group (PRDX1, annexin A1/A2). The EBC analysis by HPLC-MS/MS can be used for identification of potential protein markers specific for inflammatory pulmonary diseases of infective origin (pneumonia) as well as for non-infectious diseases such as chronic obstructive pulmonary disease (COPD).
Keywords: exhaled breath condensate; mass spectrometry; proteomics; chronic obstructive pulmonary disease (COPD); pneumonia

The role of oxidative protein modification and the glutathione system in modulation of the redox status of breast epithelial cells by E. A. Stepovaya; E. V. Shakhristova; N. V. Ryazantseva; O. L. Nosareva; V. D. Yakushina; A. I. Nosova; V. S. Gulaya; E. A. Stepanova; R. I. Chil’chigashev; V. V. Novitsky (235-239).
The effects of N-ethylmaleimide (NEM) and 1,4-dithioerythritol (DTE) on the level of oxidative modification of proteins, the state of glutathione and thioredoxin systems and the cellular redox status have been investigated in HBL-100 cells (breast epithelial cells). Breast epithelial cells cultivated in the presence of NEM were characterized by the decreased redox status, increased glutathione reductase activity, and increased concentrations of products of irreversible oxidative modification of proteins and amino acids. Cell cultivation in the presence of DTE shifted the redox status towards reduction processes and increased reversible protein modification by glutathionylation. The proposed model of intracellular redox modulation may be used in the development of new therapeutic approaches to treat diseases accompanied by impaired redox homeostasis (e.g. oncologic, inflammatory, cardiovascular and neurodegenerative disease).
Keywords: oxidative stress; cell redox status; protein oxidative modification; breast epithelial cells

The aim of this study was to determine reference values of matrix metalloproteinases (MMPs) MMP-1, MMP-2, MMP-9 and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in the amniotic fluid at the first stage of labor in physiological pregnancy. Eighty nine women at the first stage of term labor have been examined. Samples of amniotic fluid were taken at the first period of labor by vaginal amniotomy. Concentrations of MMP-1, MMP-2, MMP-9, and TIMP-1 have been investigated in amniotic fluid samples by ELISA kits. The following normal concentration ranges for MMP-1, MMP-2, MMP-9, TIMP-1, and ratios of concentrations of MMPs and TIMP-1 (MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-9/TIMP-1) have been determined for amniotic fluid samples obtained during the first period of labor in physiological pregnancy. These included: MMP-1: 5.1–16.8 pg/mg of protein; MMP-2: 238.3–374.1 pg/mg of protein; MMP-9: 66.1–113.3 pg/mg of protein, TIMP-1: 4.7–13.6 pg/mg of protein, MMP-1/TIMP-1 ratio: 0.1–2.2, MMP-2/TIMP-1 ratio: 19.9–55.7; MMP-9/TIMP-1 ratio: 4.2–17.2.
Keywords: amniotic fluid; matrix metalloproteinases; labor

Urea formation by postoperative liver by P. N. Savilov (243-245).
The effect of liver left lobe resection (LR, 15–20% of the organ weight) on hepatic urea formation was investigated in 84 albino rats. The objects used for analysis of urea content included: operated left lobe (LLL), non-operated middle (MLL) lobe of the liver, blood (aorta, v. hepatica, v. porta), and choledochal bile. Arginase activity was examined in liver homogenate. On the day 3 and day 7 after resection reduced arginase activity was detected. LR caused a decrease in urea content in v. hepatica, but increased urea content in the arterial blood and v. porta. The increase in bile urea on day 7 was then changed for its decrease observed on day 14 of the postoperative period. The urea content in the liver on day 3 after LR was below the norm, while on days 7 and 14 it became normal. Results of this study suggest impaired urea formation by hepatocytes of the operated liver and activation of extrahepatic mechanisms of urea formation from arginine.
Keywords: liver resection; urea; arginase

Activation of caspases in lymphocytes of patients with borderline personality disorders by E. S. Gerasimovich; A. A. Yakovlev; T. A. Druzhkova; M. N. Grishkina; A. B. Guekht; N. V. Gulyaeva (246-250).
Activities of caspases involved in cell death have been investigated in lymphocytes of patients with borderline personality disorders (BPD). In the group of patients with organic mental disorders (OMD) caspase activity decreased in a depressive disorder and increased in an anxiety disorder, thus suggesting activation of apoptosis in lymphocytes of patients with the anxiety disorder. Caspase activities differed in lymphocytes of OMD patients with the depressive disorder and a recurrent depressive disorder. In BPD patients with OMD lymphocyte caspase activity was higher thus indicating activation of apoptosis in these cells. Results can be used for specification of mechanisms of pathogenesis of mental disorders and search for new markers of these diseases.
Keywords: depression; lymphocytes; apoptosis; caspases; plasma peptides; borderline personality disorders

The effect of diet supplementation with polyunsaturated fatty acids (PUFAs) used at different ratios of ω-6/ω-3 on the content of primary (diene conjugates, DC; triene conjugates, TC), secondary (ketodienes, CD; coupled trienes, CT; TBA-active products) and terminal (Schiff bases) lipid peroxidation products (LPO) and generation of superoxide anion-radical was studied in rat cardiac mitochondrial fraction. The cardiac mitochondrial fraction of rats kept on a diet with a high content of ω-6 and ω-3 PUFAs for eight weeks was characterized by increased content the primary, secondary and final LPO and a higher rate of superoxide radical formation. In the case of diet supplementation with ω-6 and ω-3 PUFAs used at the ratio of 4: 1, the leading factor determining LPO intensity in the cardiac mitochondrial fraction is a species PUFA composition rather than the degree of saturation.
Keywords: lipid peroxidation; superoxide anion-radical; polyunsaturated fatty acids; mitochondrial fraction; heart

The effect of xenobiotics on microRNA expression in the rat liver by L. F. Gulyaeva; M. D. Chanyshev; S. K. Kolmykov; D. S. Ushakov; S. S. Nechkin (258-263).
The effect of xenobiotics on microRNA expression in the rat liver has been investigated. Based on results of bioinformatics analysis several microRNAs that can interact with 3'-untranslated regions of cytochrome P450 (CYP) mRNAs have been selected. These included three microRNAs (miR-21, miR-221, miR-222) for CYP1A1 mRNA as a putative target and two microRNAs (miR-143, miR-152) for CYP2B1 mRNA as a putative target. Using the RT-PCR method, expression levels of these microRNAs have been detected in the liver of rats treated with inducers of CYP1A and CYP2B, benzo(a)pyrene (BP), phenobarbital (PB), and DDT. In rats treated with both BP and DDT the hepatic content of miR-21, miR-221 and miR-222 was 2−3 times lower than in the control animals, while ethoxyresorufin-O-deethylase (EROD) activity of CYP1A1 demonstrated a 5.5−8.7-fold increase. In PB-treated rats miR-143 expression remained unchanged, the level of miR-152 increased 2-fold, while pentoxyresorufin-O-deetylase (PROD) activity of CYP2B increased 10.5-fold. In the liver of DDT-treated rats PROD activity demonstrated a 20.8-fold increase; expression of miR-143 increased 2-fold, and miR-152 expression remained unchanged. Bioinformatics analysis of putative miR-target interactions showed that the selected microRNAs can potentially bind such target as AhR, ESR1, GR, CCND1, PTEN mRNAs. Thus, the expression profile of miR-21, miR-221, miR-222, miR-143, miR-152 may vary in dependence on the CYP inducer used. Analysis in silico has shown that besides genes encoding CYP1A/2B other genes including those involved in hormonal carcinogenesis should be considered as potential targets of the investigated microRNAs.
Keywords: benzo(a)pyrene; phenobarbital; DDT; induction; cytochrome P450; microRNA

The effect of antioxidants on in vivo and in vitro methemoglobin formation in erythrocytes of patients with Parkinson’s disease by M. G. Makletsova; G. T. Rikhireva; V. V. Poleshuk; K. V. Gryakalov; S. L. Timerbaeva; T. N. Fedorova (264-268).
Methemoglobin formation was examined in erythrocytes of 16 patients with Parkinson’s disease (PD) (stage 3–4 by the Hoehn and Yahr scale). The patients receiving levodopa-containing drugs (madopar, nakom) were also treated with intramuscular injections of mexidol (daily dose 100 mg/day) for 14 days. Control group included 12 clinically healthy persons. The erythrocyte methemoglobin content was determined by electronic paramagnetic resonance (EPR) using the EPR signal intensity with the g-factor 6.0. The methemoglobin content was significantly higher in erythrocytes of PD patients than in healthy donors. The complex therapy with mexidol normalized the methemoglobin content in erythrocytes of PD patients. Incubation in vitro of erythrocytes of donors and PD patients with acrolein increased the methemoglobin content, while incubation with carnosine normalized the methemoglobin content in erythrocytes of PD patients. Prophylactic (i.e. before acrolein addition) and therapeutic administration of carnosine to the incubation system with acrolein decreased the methemoglobin content to its initial level. Results of this study suggest that inclusion of the antioxidants mexidol and carnosine in the scheme of basic therapy of PD may reduce side effects associated with methemoglobinemia.
Keywords: methemoglobin; Parkinson’s disease; antioxidants; mexidol; carnosine; acrolein

Epigenetic factors in atherogenesis: MicroRNA by A. V. Smirnova; V. N. Sukhorukov; V. P. Karagodin; A. N. Orekhov (269-275).
MicroRNAs (miRNAs) are small (~22 nucleotides in length) noncoding RNA sequences regulating gene expression at the posttranscriptional level. MicroRNAs bind complementarily to certain mRNA and cause gene silencing. miRNAs are involved in plaque formation and rapture, regulation of cholesterol metabolism, inflammatory response, cell cycle progression and proliferation, platelet activation; they also influence functions of endothelial and vascular smooth muscle cells (VSMC). This suggests their important roles in initiation and progression of atherosclerosis. MicroRNAs play a key role in the development of cardiovascular diseases (CVDs), including atherosclerosis. The use of antisense oligonucleotides is considered as promising technique for targeted changes in gene expression both in vitro and in vivo. In this review, we consider the role of miRNAs in progression of atherosclerosis, their use as potential biomarkers and targets in the treatment of CVDs, as well as possible application of antisense oligonucleotides.
Keywords: microRNA; atherosclerosis; antisense oligonucleotides

In MCF-7/Dox human breast carcinoma cells, down-regulation of integrin α5β1 and inhibition of epidermal growth factor receptor (EGFR) markedly reduced cell proliferation. Cell cycle analysis showed that α5β1 down-regulation resulted in cycle arrest at the S-phase, followed by a significant increase in the population of apoptotic cells (subG1 population). Inhibition of EGFR activity also caused cell cycle arrest at the S-phase but without any increase in the subG1 population. Down-regulation of α5β1 and EGFR inhibition resulted in a significant decrease of cell content of the active (phosphorylated) forms of FAK and Erk protein kinases. The data obtained suggest that α5β1 integrin is implicated in cell growth control via inhibition of apoptotic cell death and through EGFR activation.
Keywords: integrins; growth factors; cell proliferation; oncogenesis; signaling; protein kinases

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD+/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deficiency. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD+/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein deficiency caused a more pronounced decrease in the activity of studied Krebs cycle NAD+-dependent dehydrogenases and a 2.2-fold increase of the mitochondrial NAD+/NADН ratio.
Keywords: alimentary protein deficiency; acetaminophen-induced hepatitis; isocitrate dehydrogenase; α-ketoglutarate dehydrogenase; malate dehydrogenase; NAD+/NADН ratio

An antitumor osteotropic agent based on tumor necrosis factor by L. R. Lebedev; E. D. Danilenko; Yu. V. Telegina; B. N. Zaitsev (287-291).
An osteotropic agent based on the human recombinant tumor necrosis factor alpha (TNF-alpha) has been designed for treatment of bone metastases. It represents a molecular construct containing yeast double- stranded ribonucleic acid (dsRNA) covered by the conjugate of polyanion dextran with TNF-alpha and bisphosphonate alendronic acid. This construct is characterized by the combination of substances possessing antitumor activity (TNF-alpha, dsRNA) and a vector molecule (bisphosphonate) providing tropism to hydroxyapatite, the main mineral component of the bone tissue matrix. The conjugation conditions were optimized and the conjugates of TNF-alpha and alendronic acid with dextran were synthesized. The molecular constructs were obtained by self-assembly, and the resultant complexes were separated by gel filtration on Sepharose CL-6B. The electrophoretic analysis has shown decreased mobility of dsRNA in the complex with the conjugate as compared to mobility of the original dsRNA. This confirms formation of the designed structures. Transmission electron microscopy confirmed the presence of particles with sizes of 30–40 nm in the drug preparation. Evaluation by the sorption/desorption method showed a higher affinity of TNF-alpha conjugates to hydroxyapatite as compared to original TNF-alpha molecules (from 1.0–1.8 mol/L vs. 0.3 mol/L of potassium phosphate buffer for desorption, respectively).
Keywords: tumor necrosis factor; alendronic acid; nanoparticles