Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.1, #4)

The synthesis of artificial genome as the basis of synthetic biology by S. P. Radko; A. P. Il’ina; N. V. Bodoev; A. I. Archakov (277-283).
Recent achievements in the whole-genome sequencing especially viral and bacterial ones together with the development of methods of bioinformatics and molecular biology, have created preconditions for transition from synthesis of genes to assembly of the whole genomes based on chemically synthesized blocks, oligonucleotides. The creation of artificial genomes and artificial cells will undoubtedly render huge influence on a deepening of knowledge on mechanisms of functioning of living systems at a cellular level, on a way of origin and evolution of life, and also on biotechnology of the future, and will generate preconditions for the further development of synthetic biology and nanobiotechnology.
Keywords: synthesis of genome; synthetic biology; nanobiotechnology

Literature data on the role of oxidative stress in aging have been summarized. There are certain links between parameters of free radical processes (intensity of generation of reactive oxygen species in mitochondria, oxidative modification of mitochondrial DNA, activity of desaturases, involved into biosynthesis of polyunsaturated C20 and C22 fatty acids) with life span. The review highlights the role of oxidative stress as on of pathogenic factors of numerous diseases including various neurodegenerative disorders. Special attention is paid to oxidative modification of proteins as one of early and reliable markers of tissue injury in free radical pathology. Oxidative destruction of proteins plays a major role in etiology of such neurodegenerative diseases as Alzheimer’s and Parkinson’s diseases. Oxidative stress and the stress related protein aggregation are considered as the pathogenic link in the development of familiar amyotrophic lateral sclerosis. Oxidative modification of proteins is associated with the development of cataract. The age-and pathology-related increases in the content of oxidized proteins in tissues is assessed as an early and specific parameter of oxidative stress.
Keywords: oxidative stress; reactive oxygen species; oxidative modification of proteins; aging; neurodegenerative diseases

Blood deoxyribonuclease activity in health and diseases by A. V. Cherepanova; S. N. Tamkovich; V. V. Vlassov; P. P. Laktionov (299-304).
The review summarizes literature data on (deoxy)ribonuclease activity in blood of healthy donors and patients with diseases. Special attention is paid to methodological aspects of the analysis of deoxyribonuclease activity and factors, which interfere with enzyme activity in blood.
Keywords: blood deoxyribonucleases; malignant diseases; autoimmune diseases

Nonsteroidal anti-inflammatory drugs: I. A study of “structure-efficacy of the anti-inflammatory effect relationship activity” by V. R. Khayrullina; A. D. Mukhametov; L. A. Tjurina; G. G. Garifullina; A. Ya. Gerchikov; Ph. S. Zarudiy (305-312).
Using the computer system SARD-21 (Structure Activity Relationship & Design) structural features typical for the high-and low-effective nonsteroidal anti-inflammatory drugs (NSAIDs) have been recognized and the influence of these features on the anti-inflammatory properties have been evaluated. This information has been used for generation of the model for prediction of anti-inflammatory effectiveness of pharmaceutical preparations with 76% and 81% levels of recognition by two methods. The recognized structural parameters may be successfully used for creation of new highly effective NSAIDs, and also for modification of structures of known NSAIDs for the increase of effectiveness of their anti-inflammatory action.
Keywords: Nonsteroidal anti-inflammatory drugs; theory of shape recognition; structure-activity; cyclooxygenase

Mechanisms of attenuation of ischemic heart injury by means of a modified reperfusion by O. I. Pisarenko; I. M. Studneva; V. S. Shulzhenko; A. A. Timoshin (313-318).
Mechanisms of attenuation of membrane injury and metabolic impairments in postischemic cardiomyocytes have been studied on a model of ischemic and reperfusion stress of rat heart using a modified early reperfusion. Optimization of the reperfusion infusate composition augmented recovery of cardiac pump and contractile function. This was accompanied by reduced release of lactate dehydrogenase activity and systems generating short-living reactive oxygen species into myocardial effluent and was associated with more efficient oxidative metabolism recovery and decreased losses of intracellular total creatine and amino acids pools. The results indicate perspectives of postischemic functional and metabolic myocardial injury correction by means of the controlled reperfusion.
Keywords: reperfusion of the heart; high energy phosphates; amino acids; active oxygen forms; myocyte membranes

Combined inhibiting action of new salicylic acid derivatives and α-tocopherol on oxidation of methyl oleate by M. G. Perevozkina; A. A. Kudryavtsev; N. U. Tretyakov; N. M. Storozhok (319-326).
The inhibitory effects of compositions of α-tocopherol (α-TP) and salicylic acid derivatives on the process of initiated oxidation of methyl oleate have been investigated. α-TP and the salicylic acid derivatives exhibited the synergistic effect, which was demonstrated by the methods of UV-spectroscopy and high-performance liquid chromatography (HPLC). Kinetics of α-TP utilization during methyl oleate oxidation was investigated under conditions of its independent use as well as using its binary mixture with the synthetic antioxidants.
Keywords: antioxidants; synergists; α-tocopherol; peroxide oxidation; antiradical activity; salicylic acid derivatives

Nanoelectrochemistry of cytochrome P450s: Direct electron transfer and electrocatalysis by V. V. Shumyantseva; T. V. Bulko; Yu. O. Rudakov; G. P. Kuznetsova; N. F. Samenkova; A. V. Lisitsa; I. I. Karuzina; A. I. Archakov (327-333).
Direct electron transfer has been demonstrated between cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14α-demethylase (CYP51MT) and screen printed graphite electrodes, modified by gold nanoparticles and didodecyldimethyl ammonium bromide (DDAB). The proposed method for preparation of enzymatic nanostructured electrodes may be used for electrodetection of this hemoprotein provided that 2–200 pmol P450 per electrode has been adsorbed.Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, lanosterol) and inhibitor ketoconazole were analyzed using cyclic voltammetry (CV), square wave (SWV) or differential pulse (DPV) voltammetry, and amperometry.
Keywords: cytochrome P450 2B4; P450 1A2; sterol 14α-demethylase from Mycobacterium tuberculosis (CYP51MT, P45014DM); lanosterol; ketoconazole; screen printed electrodes; bioelectrochemistry; gold nanoparticles; nanostructured electrodes

The influence of Zn(II) and Mn(II) on catalytic activity of C. albicans aspartic proteinases by M. P. Kutyreva; R. R. Galimzanova; N. A. Ulakhovich; N. I. Glushko (334-341).
The interaction of secreted aspartic proteinases from C. albicans (C. albicans SAP) with ZnCl2 and MnCl2 has been studied. Logarithms of stability constant from the data of electronic spectroscopy were calculated for the complexes of SAP with Zn (II) (SAP-ZnII, logβ = 4.73 ± 0.20) and with Mn(II) (SAP-MnII, logβ = 7.02 ± 0.20). The composition and maximum accumulation of complexes in solution were calculated. The optimal conditions of hydrolysis of the substrate, HAS (human serum albumin) in the presence of C. albicans SAP-MnII and SAP-ZnII proteinases were determined. These were: [HSA] = 1 mg/ml, [SAP] = 2.33 μM, pH = 4.5, incubation time of 25 min. The activity of C. albicans SAP in the presence of different concentrations of ZnCl2 and MnCl2 was evaluated under optimal conditions of enzymatic hydrolysis. For the first time the activating action of 0.5 μM ZnCl2 on catalytic activity of C. albicans SAP proteinase has been demonstrated. The maximal rate of enzymatic reaction (V max), the Michaelis constant (K m ) and constants of effects in the presence and in the absence of the effector, ZnCl2, were calculated. The albuminase activity of C. albicans pathogenic strains of different localization was evaluated in the presence and the absence of the effector of ZnCl2.
Keywords: proteinases of Candida albicans ; enzymatic catalysts; effectors; kinetic parameters

Matrix metalloproteinases (MMPs) play a critical role in tumor development and invasion. The aim of this study was to elucidate peculiarity of expression of interstitial collagenase (MMP-1) and its endogenous regulators during oncogenic transformation of fibroblasts by HPV-16 E7 gene. Papilloma virus types 16 and 18 are etiological factor of cervical cancer. We have studied expression of MT1-MMP, MMP-1, tissue inhibitor of these proteases, TIMP-1, and urokinase-like plasminogen activator (uPA), activating MMP-1 via plasmin. The study was carried out using fibroblasts immortalized by LT gene (IF) and transformed by E7 gene of HPV-16 fibroblasts (TF). Primary culture of Fisher rat embryo fibroblasts was used as a control (PF). mRNA expression, and enzymatic activity were studied by RT-PCR and by hydrolysis of fluorogenic type I collagen, respectively. Cell transformation was accompanied by: (a) 2–3 fold induction of MT1-MMP mRNA expression vs PF; (b) the decrease in mRNA level of TIMP-1 (1,5–2 fold); c) unchanged uPA expression. Cell immortalization is accompanied by: (a) the increase of MT1-MMP expression (1,5–2 fold); (b) unchanged TIMP-1 expression; (c) the increase of uPA expression (2–4 fold) vs PF and TF. MMP secreted activity and activity in lysates of TF increased but level of free endogenous MMP inhibitors decreased vs IF. Data on gene expression are consistent with enzymatic data on the collagenolytic activity. These results suggest changes in enzyme/inhibitor/activator ratio both TF and IF and significant enhancement of the destructive potential of the TF.
Keywords: matrix metalloproteinases; MMP-1; MT1-MMP; TIMP-1; uPA; HPV-16

Tumor cell derived matrix metalloproteinases are a family of enzymes associated with the tumor invasion and metastasis. Extracellular matrix metalloproteinases inducer (EMMPRIN) stimulates synthesis of gelatinase A (MMP-2) in peritoneal fibroblasts. In the present study the role of MMP-2 and EMMPRIN in the progression of breast cancer has been investigated. Gelatinase-A and EMMPRIN were analyzed in benign as well as in stage II and stage III breast cancer tissue samples by gelatin zymography assay, immunoprecipation analysis and Western blot analysis with a monoclonal primary antibody specific for EMMPRIN. Our results showed over expression of EMMPRIN in advanced stages of breast cancer tissues compared with benign tumor tissue samples. The expression of MMP-2, the active and latent forms of the enzyme increased with tumor progression from Stage II to Stage III of breast cancer and it was not expressed in benign tissues. The expression MMP-2 correlates with tumor progression. This observation obviously indicates that EMMPRIN and MMP-2 are the major determinants of malignancy in cancers.
Keywords: EMMPRIN; MMP-2; tumor invasion; breast cancer; Western blot

The development of a new adjuvant lipid-saponin complex and its use at experimental immunization by bacterial antigen by A. V. Tsybulsky; N. M. Sanina; I. A. Lee; A. M. Popov; E. Ya. Kostetsky; O. Yu. Portnyagina; V. L. Shnyrov (353-358).
Immunostimulating complexes (ISCOM) have been modified by replacement of structural components of ISCOM; phosphatidylcholine has been replaced, by glycolipid of monogalactosyldiacylglycerol from sea macrophytes, saponin QuilA has been replaced by triterpene glycoside, Cucumarioside A2-2. The resultant complex includes morphological structures of two types: the ISCOM-like structures with the characteristic morphology and sizes and also the tubular structures with diameter of about 40 nm and length of 150–400 nm. We have defined these structures as TI-complexes. These TI-complexes exhibit considerably lower toxicity than ISCOM. They may include an amphiphilic protein antigen and provide immunoadjuvant effect during experimental vaccination. Under conditions of the experimental immunization of mice with a weak immunogen (subunit membrane pore protein from Y. pseudotuberculosis), TI-complexes with antigen provided stronger humoral immune response to antigen than the complexes of porin with classical ISCOM, liposomes and Freund’s adjuvant. Thus, the TI-complexes represent a new type of adjuvant carriers for antigens.
Keywords: adjuvant; ISCOM; triterpene glycoside; monogalactosyldiacylglycerol (MGDG); vaccine; porin

Biochemical and photometric studies of modification of collagen structure induced by UV irradiation by G. A. Rebrova; V. K. Vasilevsky; L. B. Rebrov; L. A. Osipova; V. A. Bykov (359-364).
The influence of UV irradiation (270–380 nm) on the biochemical, fluorescence and colorimetric properties of collagen was studied. The long-term UV irradiation (120 h) was accompanied by the increase of the structural stability of collagen to specific and nonspecific proteolytic enzymes, by formation of new additional fluorophore containing compounds, by the increased amount of carbonyl groups in the collagen, and by significant changes in the distribution pattern of products of alkaline hydrolysis during gel chromatography. The coordinates of color of the collagen films have been also changed. These changes of collagen suggest that UV irradiation induces photomodification and photooxidation processes in collagen.
Keywords: collagen; UV irradiation; photochemistry; photoaging

The aim of this study was to determine correlation between parameters of proteolysis and phenotypes of α1-proteinase inhibitor in blood plasma of children with duodenal ulcer. Activation of kininogenesis, pepsin-and trypsin like proteinases was accompanied by the decrease in activity of α2-macroglobulin and the increase in activity of acid-stable inhibitors. Three subtypes of normal phenotype of α1-proteinase inhibitor (M1M3, M1M1, M2M2) were determined.Activation of proteolysis was more pronounced in the individuals with subtype M2M2. In M2M2 activity of α1-proteinase inhibitor was two times lower than in control, in M1M1 it insignificantly differed from control group and in the M1M3 subtype it was 1.9 times higher than in control. Low activity of α1-proteinase inhibitor seen at the M2M2 subtype was accompanied by higher activity of acid-stable inhibitors; this may be regarded as the protective compensatory reaction of the body. Determination of the α1-proteinase inhibitor phenotypes may be used for evaluation of a state of proteolysis and as a basis for employment of polyvalent proteinase inhibitors for therapy of ulcer.
Keywords: proteinases; phenotypes of α1-proteinase inhibitor; acid-stable inhibitors; α2-macroglobuline; duodenal ulcer

Biochemical and immunological markers of insufficiency of blood circulation in children with cardiomyopathies by T. V. Bershova; Yu. V. Shmatkova; A. P. Ivanov; M. I. Bakanov; E. N. Basargina; R. B. Zurabova; S. V. Monaenkova; Yu. V. Gerasimova (370-376).
Complex clinical-laboratory investigation of children with congestive heart failure developed on the basis of dilated cardiomyopathy and hypertrophic cardiomyopathy has been carried out. The development of congestive heart failure in children with cardiomyopathy was accompanied by changes in activity of creatine phosphokinase, MB isoform of creatine phosphokinase, and also blood serum lactate, neopterin, TNF-α, interleukin-2 (IL-2), interleukin-6 (IL-6), and the soluble receptors for IL-2 and IL-6 (sIL-2R, sIL-6R).The energy deficit in patients with congestive heart failure is associated with pronounced impairments of expression of neopterin, TNF-α, IL-2, IL-6 and their receptors. The role of cytokines in formation of dysregulation processes is analyzed at the level of intercellular and organ local interactions. A cascade of multiple biochemical and molecular processes including impairments of membrane integrity and ion transport, apoptosis, proteolysis followed by fibrosis of myocardium finally cause myocardial remodeling, the development, chronization and progression of congestive heart failure in children with cardiomyopathy.
Keywords: congestive heart failure; hypoxia; cardiomyopathies; cytokines; soluble receptors of cytokines; neopterin