Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.6, #3)

Integrins as a potential target for targeted anticancer therapy by A. E. Berman; N. I. Kozlova; G. E. Morozevich (205-210).
The review briefly summarizes information of structure of integrins and their involvement in the development and malignant progression of tumors. Special attention is paid to approaches based on modification of functional properties of integrins that prevent/antagonize tumor growth and progression; these approaches developed in modern experimental biology have certain perspective in clinical application.
Keywords: structure and function of integrins; integrin-specific immune preparations; peptide-based integrin antagonists; anticancer therapy

Identification of cancer stem cells-associated “side population” by flow cytometry with a violet laser by A. Yu. Lupatov; A. M. Gisina; P. A. Karalkin; K. N. Yarygin (211-217).
The possibility of identification of a “side population” of cancer stem cells in solid tumors by a flow cytometer equipped with a 405 nm violet laser has been investigated. Ovarian cancer (Skov-3) and colorectal cancer (Colo 320) cell lines formed the “side population” after vital staining with Hoechst 33342. Analysis of cells isolated from the tumor tissue of malignant melanoma and colorectal cancer, also revealed the “side population” characterized by increased fluorescent dye exclusion. The percentage of melanoma cells included in the “side population” was the same as that of cells co-expressing the cancer stem cells markers CD34 and CD44. However, the colon cancer “side population” was significantly smaller than the minor populations of colon cancer cells identified by either CD133 expression or exclusion of Rhodamine 123 exclusion.
Keywords: cancer stem cells; “side population”; flow cytometry; malignant melanoma; colorectal adenocarcinoma

Oligomeric state investigation of flavocytochrome CYP102A1 using AFM with standard and supersharp probes by Yu. D. Ivanov; N. S. Bukharina; P. A. Frantsuzov; T. O. Pleshakova; N. V. Krohin; S. L. Kanashenko; A. I. Archakov (218-224).
Atomic force microscopy with two types of probes—standard (radius of curvature R ∼ 10 nm) and supersharp (R ∼ 2 nm)—was used to determine an oligomeric state of CYP102A1. Using the standard probes CYP102A1 images were obtained in liquid, air and vacuum environments, and a CYP102A1 monomer: oligomer ratio α ≈ 1 was also determined. However, the use of standard probes did not allow to resolve structures of these oligomers. Using the supersharp probes it was possible to determine not only the monomer: oligomer ratio, but also to evaluate the dimer: trimer: tetramer ratio in vacuum. Thus, the ratio α for CYP102A1 in liquid can be determined by the standard probes in liquid, air, and vacuum, while oligomeric states of this protein can be specified by using the supersharp probes in vacuum.
Keywords: atomic force microscopy; cytochrome P450 CYP102A1; oligomeric state

The effect of a single and multiple intravenous injections of a nanosized magnetite suspension on total prooxidant and antioxidant activity of blood plasma has been investigated by the method of luminoldependent chemoluminescence. Nanoparticles of magnetite exhibited dose-dependent prooxidant properties due to their iron atoms and cause a compensatory activation of antioxidant systems in the rat blood plasma. After a single intravenous administration of magnetite the studied parameters of blood plasma returned to the normal level by the end of the experiment due to elimination of the nanoparticles from the body. In the case of multiple administration of the magnetite suspension the dose-dependent changes in the pro- and antioxidant plasma activity persisted during the whole experiment. Accumulation of magnetite particles in the cells of the rat mononuclear phagocytic system, liver, lungs and kidneys is associated with hemodynamic disorders, local dystrophic and necrotic changes in the parenchyma of these organs. After a single intravenous injection nanoparticles of magnetite are detected in the rat organs for 40 days, but their number decreases by the end of the experiment.
Keywords: nanomagnetite; blood plasma; prooxidant and antioxidant activity

The effect of melatonin on the antioxidant status of rats with type II diabetes mellitus by A. A. Agarkov; T. N. Popova; L. V. Matasova (231-236).
The effect of melatonin on the intensity of free radical processes and activities of superoxide dismutase (SOD, EC and catalase (EC has been investigated in liver and blood serum of rats with type II diabetes mellitus. According to results of this study the development of diabetes was accompanied by the increase in biochemiluminescence parameters and the enzyme activities studied. Melatonin administration changed the parameters studied towards control values and this is obviously associated with realization of the antioxidant potential of this hormone.
Keywords: type II diabetes mellitus; biochemiluminescence; superoxide dismutase; catalase; melatonin

This study was performed to investigate the effects of low power laser radiation in blue (441.2 nm), green (532.5 nm) and red (632.8 nm) wavelength ranges on free radical processes in experimental endotoxic shock in rats. The experimental model was induced by intraperitoneal injection of lipopolysaccharide B (25 mg/kg) (LPS). Functional activity of blood leukocytes was evaluated by the method of luminol-dependent chemiluminescence, plasma superoxide dismutase activity was determined by the nitro blue tetrazolium assay, intensity of lipid peroxidation in erythrocyte membranes was estimated by cis-parinaric acid fluorescence. It was found that the low power laser radiation significantly influenced all investigated processes, in LPS-treated and control animals. The most pronounced effects were observed in all groups of LPS-treated animals, in which the laser radiation increased all investigated parameters. At the radiation dose 0.75 J/cm2 green laser was the most effective, while at the dose of 1.5 J/cm2 both green and red lasers produced potent effects. Possible mechanisms of the observed phenomena are discussed.
Keywords: endotoxic shock; low power laser radiation; chemiluminescence; reactive oxygen species; leukocytes; superoxide dismutase activity; lipid peroxidation

Matrix metalloproteinases (MMP) play a key role in development of tumor invasion and metastasis. The aim of the work was to elucidate peculiarities of expression of MMP-1, MMP-2, MMP-9 and regulators of their activity: plasminogen activator uPA and tissue inhibitors of MMPs TIMP-1 and TIMP-2 in human cell lines of squamous cell carcinoma (SCC).The comparative study on MMPs expression was carried out on SCC cell lines differed by types of human papillomavirus (HPV) (HPV-16 and HPV-18): SiHa and Caski cells had ÍĐV16, Hela, Ñ4-1 — HPV18. The Ñ33Ã cell line lacking HPV copies was used as control. The study of MMP expression included estimation of mRNA expression using the RT-PCR method and determination of collagenolytic activity by hydrolysis of fluorogenic type 1 collagen and also by the zymography method. It was shown that: 1. In both types of cell lines, the MMP-1 expression demonstrated a significant (from 2 to 8-fold) increase, being the most pronounced in HPV18 cell lines. The only exception was the SiHa cell line, in which decreased expression of this enzyme was observed. MMP-2 expression remained at the control level in both types of cell lines. 2. Expression of inhibitors was within the control level. The only exception was found in the C4-1 line where TIMP-1 and TIMP-2 expression increased by 1.7–2.6-fold, respectively. Increased (2–4.5-fold) expression of uPA was found in all cell lines except SiHa where it decreased by 20%. 3. Collagenolytic activity in the Caski and Hela cell lines was 2–3 times higher than in control, while the activity in the SiHa cell line was comparable with the control level. The study gelatinolytic activity as well as the data on mRNA revealed only the presence of MMP-2, but not MMP-9 in all cervical carcinoma cell lines. The data obtained suggest significant impair- ments of the ratio enzyme/inhibitor/activator in the transformed cell lines; these impairments are mainly associated with increased expression of MMP-1 and its activator, whereas expression of MMP-2 and its inhibitors remains basically virtually unchanged. This results in the increase of the destructive potential of cervical carcinoma cell lines.
Keywords: matrix metalloproteinases (MMP) — MMP-1,-2,-9; tissue inhibitors of MMPs; cervical carcinoma cell lines

The addition of nitro blue tetrazolium (NBT) into the reaction of adrenaline autooxidation allows direct identification of superoxide anion formation (O 2 −⊙ ) as well as demonstration of kinetics of their accumulation in this superoxide-generating system. The kinetics of adrenochrome and O 2 −⊙ formation has been compared under the same conditions. Three possible approaches to the use of the adrenaline autooxidation reaction for the determination of superoxide dismutase activity (SOD) and revealing antioxidant properties of various compounds are discussed. Two of these approaches have been described previously: the spectro-photometric method of registration of adrenochrome, an end product of adrenaline autooxidation, at 347 nm (Sirota, 1999) and the polarographic method, which measures oxygen consumption used for O 2 −⊙ formation (Sirota, 2011). Here, a novel approach to this problem is presented; it is based on spectrophotometric determination of O 2 −⊙ using NBT. The employment of this approach results in a significant decrease of the pH value of carbonate buffer from 10.5 to 9.7 and a 4-fold decrease in the amounts of added adrenaline, thus creating milder conditions for the revealing and investigation of antioxidant properties of materials being examined.
Keywords: adrenaline; nitro blue tetrazolium; adrenochrome; superoxide; oxygen; superoxide dismutase

Arginine and lysine as products of basic carboxypeptidase activity associated with fibrinolysis by A. A. Zhloba; T. F. Subbotina; D. S. Lupan; V. A. Bogova; O. A. Kusheleva (261-265).
Blood carboxypeptidases play an important role in the regulation of fibrinolysis. We have proposed here the method for the assay of blood carboxypeptidase activity associated with coagulation/fibrinolysis using the natural substrate fibrin and the detection of basic amino acids arginine and lysine as products under conditions closely resembling those in vivo. Plasma samples from 15 patients with arterial hypertension have been investigated. Coagulation and subsequent fibrinolysis were initiated by addition of standard doses of thrombin and tissue plasminogen activator, respectively. Arginine and lysine concentrations before, during, and after completion of fibrinolysis were determined using HPLC. The parameters of fibrinolysis were evaluated by the clot turbidity assay. The coagulation/fibrinolysis cycle was accompanied by a significant increase in concentrations of arginine and lysine in the incubation mixture by 101 and 81%, respectively. The duration of fibrinolysis initiation significantly correlated with the degree of increase of these amino acids: r S = −0.733 and −0.761 for arginine and lysine, respectively (p < 0.05). Arginine generation had two maximums: one in the beginning of clot lysis and another one at the end of the lysis, whereas the lysine release occurred mainly in the middle of fibrinolysis. Thus, the carboxypeptidase activity associated with fibrinolysis can be considered as a local source of the essential amino acids originated from fibrin clot degradation products.
Keywords: fibrinolysis; basic carboxypeptidases; arginine; lysine

Activation of ganglioside GM3 biosynthesis in human blood mononuclear cells in atherosclerosis by E. V. Gracheva; N. N. Samovilova; N. K. Golovanova; G. F. Piksina; V. S. Shishkina; N. V. Prokazova (266-272).
Using blood monocytes and lymphocytes from atherosclerotic patients and healthy subjects we have investigated a role of ganglioside GM3 in monocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC). The results showed that activity of GM3 synthase and cellular levels of ganglioside GM3 in blood mononuclear cells from atherosclerotic patients were several-fold higher than those from healthy subjects. In monocytes the activity of GM3 synthase was one order of magnitude higher than in lymphocytes from both groups studied; this suggests the major contribution of monocytes to enhanced biosynthesis and levels of GM3 in mononuclear cells in atherosclerosis. Enrichment of monocytes from healthy subjects with ganglioside GM3 by their incubation in the medium containing this ganglioside increased adhesion of these monocytes to HUVEC up to the level typical for monocytes from atherosclerotic patients. In addition, an increase in CD11b integrin expression comparable to that seen in lipopolysaccharide-activated monocytes was observed. It is suggested that in atherosclerosis the enhanced cellular levels of GM3 in monocytes and lymphocytes may be an important element of cell activation that facilitates their adhesion to endothelial cells and penetration into intima.
Keywords: ganglioside GM3; GM3 synthase; blood mononuclear cells; monocytes and lymphocytes; atherosclerosis

Erythrocyte antioxidants in aging and dementia by E. A. Kosenko; L. A. Tikhonova; A. S. Pogosyan; Yu. G. Kaminsky (273-277).
Age of patients and oxidative stress in brain cells may contribute to pathogenesis of Alzheimer’s disease (AD). Erythrocytes (red blood cells, RBC) are considered as passive “reporter cells” for the oxidative status of the whole body and remain poorly investigated in AD. The aim of this study was to assess whether the antioxidant status of RBC changes in aging and AD. Blood was taken from AD and non-Alzheimer’s dementia patients, aged-matched and younger controls. The antioxidant status of RBC was evaluated in each person participated in the study by measuring levels of H2O2, organic hydroperoxides, glutathione (GSH) and glutathione disulfide (GSSG), activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase. In both aging and dementia, oxidative stress in RBC was shown to increase as evidenced by elevated concentrations of H2O2, organic hydroperoxides, decreased GSH/GSSG ratio, and decreased glutathione S-transferase activity. Decreased glutathione peroxidase activity in RBC may be considered as a new peripheral marker for Alzheimer’s disease while changes of other parameters of oxidative stress reflect age-related events.
Keywords: Alzheimer’s disease; aging; erythrocytes; oxidative stress; antioxidants

The low-molecular-weight DNA appears in blood plasma a few hours after exposure of rats to ionizing radiation, and its content correlates directly with the irradiation dose. Cloning has shown, that the lowmolecular-weight DNA is enriched with C + G pairs and features of its primary structure characterize its capacity to form rather stable nucleosomes. DNA isolated after irradiation of rats with principally different doses 8 and 100 Gy differed not only quantitatively, but also by the content of the dinucleotides CpG and CpT; this suggests that they originate from different regions of the genome. It has been shown for the first time that exposure of animals to low-frequency noise results in an increase of the content of blood plasma low-molecular-weight DNA. Strokes are characterized by the increase of this DNA during 3 days after the beginning of the acute period, and dynamics of its excretion differs in patients with ischemic and hemorrhagic forms of this disease. In the case of ischemia low-molecular-weight DNA appears in cerebrospinal fluid. In contrast to patients with non-obstructive chronic bronchitis, patients with the chronic obstructive pulmonary disease in the state of remission are characterized by decreased levels of blood plasma low-molecular-weight DNA. The clear dependence between formation and specific features of the low-molecular-weight DNA fraction in blood plasma suggests that it may serve as a universal quantitative marker of apoptosis, which differentiates basically different conditions of the body.
Keywords: low-molecular-weight DNA; ionizing radiation; nucleosomes; low-frequency noise; ischemic stroke; chronic obstructive pulmonary disease; apoptosis

The proinsulin level in blood of children with type 1 diabetes mellitus of various duration by N. Yu. Lotosh; A. A. Selishcheva; S. A. Nadorov; B. A. Badyshtov; I. E. Volkov; S. V. Saveljev (288-291).
Proinsulin content was measured in the serum of 82 children (aged from 3 to 14 years) with type 1 diabetes mellitus of various duration. Three groups of patients characterized by low (54%), normal (42%) and high (4%) levels of this prohormone were recognized. No dependence between the proinsulin level and duration of this disease term was found. The serum proinsulin level may be used as a parameter specifying pathogenesis of type 1 diabetes mellitus.
Keywords: diabetes mellitus type 1; proinsulin