Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.5, #1)

Where intestinal epithelial stem cells are localized? About molecular markers by S. Ya. Proskuryakov; A. G. Konoplyannikov; Yu. G. Verkhovskii; L. P. Ulyanova; A. F. Tsyb (1-9).
Using stem cells as an example the review considers a new history and methodology of search for stem cells (SC), found in tissues of adult Homo sapiens and Drosophila melanogaster organisms. These studies of SC resulted in several original hypotheses explaining their unusual features. Impressive progress recently achieved in this direction (2008–2010) is associated with employment of new methods of somatic recombination for long-term registration of various strains of differentiated cells, early and distant SC progeny. 1) Although anatomic localization of intestinal epithelium cells lacking marked morphological and biochemical differentiation markers (the lower third of intestinal and colon crypts) is known for about 40 years results of their experimental identification, isolation and detection of their functional characteristics still represent the subject for discussions. Particularly, it remains unclear, which SC are involved in crypt regeneration: the same as those involved into homeostatic renewal or their various subpopulations or early SC progenies acquired stem features by reprogramming? 2) In addition, most detected biochemical markers of potential SC are common for SC from other tissues of embryonic and mature organisms so it is possible to apply method developed for intestinal epithelium for their isolation. 3) Data on induction of intestinal epithelium polyps and neoplasias by mutations in genes encoding SC markers and identification of biochemical characteristics of potential SC in these tumors support the hypothesis of stem tumor cell origination from normal SC or their earliest progeny. In general, facts considered in this review may be useful for both development of optimal methods for the use of SC in cell therapy (as the source of humoral factors), regenerative medicine (as the source of differentiated cells for restoration of injured tissue), and also for targeted search of antitumor drugs (SC as the target) and preparations modifying genetic and epigenetic reactions of SC to genotoxic and stress treatments.
Keywords: stem cells; intestinal epithelium; markers

Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate)-based biopolymer systems by A. P. Bonartsev; G. A. Bonartseva; K. V. Shaitan; M. P. Kirpichnikov (10-21).
Biodegradable biopolymers attract much attention in biology and medicine due to its wide application. The present review considers a biodegradable and biocompatible polymer of bacterial origin, poly(3-hydroxybutyrate), which has wide perspectives in medicine and pharmaceutics. It highlights basic properties of biopolymer (biodegradability and biocompatibility) and also biopolymer systems: various materials, devices and compositions based on the biopolymer. Application of poly(3-hydroxybutyrate)-based biopolymer systems in medicine as surgical implants, in bioengineering as cell culture scaffolds, and in pharmacy as novel drug dosage forms and drug systems are also considered.
Keywords: biopolymer system; polyhydroxyalkanoates (PHA); poly(3-hydroxybutyrate) (PHB); biodegradation; biocompatibility

The study of DJ-1 protein in tissue specimens, cultured cells and serum of prostate cancer patients by K. V. Lisitskaya; L. S. Eremina; A. V. Ivanov; M. A. Kovalyova; V. E. Okhrits; I. Yu. Toropygin; L. I. Kovalyov; S. S. Shishkin (22-28).
Two electrophoretically distinguishable isoforms of Dj-1 protein have been identified in a proteomic study of tissue specimens obtained from patients with confirmed prostate cancer (PCa) and benign prostatic hyperplasia (BPH). Dj-1 was also found in the cell lines PC-3, DU-145, LNCaP, BPH-1, and the lowest level of Dj-1 was found in BPH-1. An immunochemical study (ELISA) of serum levels of Dj-1, Bcl-2, IGF-1 and IGFBP-3 proteins revealed statistically significant differences between these two groups of patients only for Dj-1 (p = 0.004, the Wilcoxon-Mann-Whitney test). These data suggest that Dj-1 protein is a perspective candidate biomarker for PCa.
Keywords: Dj-1; prostate cancer; two-dimensional electrophoresis; ELISA

It is known that the metabolic syndrome (MS) characterized by hypertension, dyslipidemia, glucose tolerance, and obesity leads to serious cardiovascular diseases, which still are the leading cause of mortality in developed countries. The MS distribution is growing catastrophically, but molecular mechanisms responsible for developments of complex impairments in MS still remain basically poorly investigated. Since the chronic stress plays a crucial role in MS development, in the present work a hypertensive rat strain with Inherited Stress-Induced Arterial Hypertension (ISIAH-strain) was used as a model. It was shown that the ISIAH rat strain is characterized by an increased content of triglyceride, VLDL and LDL cholesterols, a decreased content of HDL cholesterol, a high level of apolipoprotein B-100, and a decreased level of apolipoprotein A-I as compared with the control WAG rat strain. The ISIAH rat body weight and blood glucose level were higher than in WAG rats. Thus, hypertension in ISIAH rats is accompanied by dislipidemia, increased glucose level, and increased body weight, representing a whole set of symptoms typical for MS. The DNA-binding activity of the transcription factors involved into regulation of lipid and carbohydrate metabolism genes (PPAR, LXR, PXR, and CAR) was higher in ISIAH rats compared with WAG rats. A complex study of regulatory mechanisms, signaling pathways, and transcription targets mediating PPAR, LXR, PXR, and CAR effects may help better understanding of the MS pathogenesis and provide valuable information for design of complex drugs for MS treatment.
Keywords: metabolic syndrome; hypertensive rat strain ISIAH; PPAR; LXR; PXR; CAR

Expression of the renin angiotensin system genes in the kidney and heart of ISIAH hypertensive rats by L. A. Fedoseeva; M. A. Ryazanova; E. V. Antonov; G. M. Dymshits; A. L. Markel (37-43).
The content of mRNA of renin-angiotensin system (RAS) genes was measured in the kidney and heart of hypertensive ISIAH and normotensive WAG rats using the real-time PCR. A statistically significant decrease in the mRNA level of RAS genes was registered in the kidney of ISIAH rats, including Ren (by 45%), Ace (43%), AT1A (34%), COX-2 (50%). The level of myocardial expression of AT1A decreased by 28% while Ace expression increased by 80%. These results suggest reduction of renal RAS basal activity in the hypertensive ISIAH rats, and therefore this strain of rats may be referred to the group of models of low-renin hypertension. The ISIAH rats were also characterized by a two-fold increase in the connective tissue sodium concentration and also by a small (but statistically significant) increase in plasma sodium concentration (139 ± 0.3 mmol/l versus 136 ± 0.25 mmol/l in WAG rats). These results together with a tendency to a decrease of plasma aldosterone level also support existence of a classical low-renin hypertension in the ISIAH rats. It is suggested that altered function of renal ion channels represents a basis for the development of low renin hypertension in the ISIAH rats. In addition, impairments in renal system of NO synthesis may also contribute to the pathogenesis of arterial hypertension in the ISIAH rats.
Keywords: arterial hypertension; renin-angiotensin system; ISIAH rat strain; real-time PCR

The effect of an endocannabinoid congener, N-stearoylethanolamine (NSE), on the content of plasma and liver pools of free amino acids (AA) was studied in burned rats. After application of a thermal skin burn (stage III) animals perorally received an aqueous suspension of NSE (10 mg/kg of body weight) during 7 days or were treated with the aqueous NSE suspension (10 mg/ml) applied onto the burn wound, or received a combined treatment. It has been originally demonstrated for the first time, that the treatment of burned rats with NSE prevented the decrease in total AA concentration in blood plasma and the increase in hepatic AA concentration due to modulation in concentrations of glycogenic AA. In burned animals the ratio of plasma and liver homogenate Phe/Tyr and Gly/Val increased while the Fischer ratio (Ile+Leu+Val/Phe+Tyr) decreased, and after the treatment with NSE these parameters remained at the level of intact animals. These data demonstrate that NSE possesses adaptogenic properties, and it is involved in the organism response to the burn. This prevents changes in blood plasma and hepatic pools of free AA of NSE-treated rats with the burn wound compared with untreated animals.
Keywords: N-stearoylethanolamine; free amino acids; burn

The study of cytokine content and ganglioside metabolism in experimental brain edema by A. V. Zakaryan; G. S. Kazaryan; G. V. Zakaryan; M. M. Melkonyan; L. M. Hovsepyan (51-54).
A blood cytokine profile and also the brain content of lipid peroxidation (LPO) products and gangliosides were investigated in rats with experimental brain edema. The development of brain edema was accompanied by the increase in pro-inflammatory and a decrease in anti-inflammatory cytokine content. In parallel, accumulation of LPO products (conjugated dienes, hydroperoxides, and malondialdehyde) was observed. The study of ganglioside content under conditions of experimental brain edema revealed a decrease of their hydrolytic degradation product, sphingosine.
Keywords: brain edema; cytokines; gangliosides; sphingosine

Screening of potential substrates or inhibitors of cytochrome P450 17A1 (CYP17A1) by electrochemical methods by V. V. Shumyantseva; T. V. Bulko; A. Yu. Misharin; A. I. Archakov (55-59).
The electrochemical redox reactions of the recombinant form of human cytochrome P450 17A1 (CYP17A1) were investigated. The hemoprotein was immobilized on the electrode modified with a biocompatible nanocomposite material based on the membrane-like synthetic surfactant didodecyldimethylammonium bromide (DDAB) and gold nanoparticles. Analytical characteristics of DDAB/Au/CYP17A1 electrodes were investigated using cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Analysis of electrochemical behavior of cytochrome P450 17A1 was performed in the presence of a natural substrate, pregnenolone (1), known inhibitor, ketoconazole (2), and in the presence of synthetic derivatives of pregnenolone: acetyl pregnenolone (3), cyclopregnenolone (4), and tetrabromo-pregnenolone (5). Ketoconazole, the azole inhibitor of cytochromes P450, blocked catalytic current in the presence of the substrate, pregnenolone (1). Compounds 3–5 did not demonstrate substrate properties towards the electrode/CYP17A1 system. Compound 3 did not influence catalytic activity with pregnenolone, whereas compounds 4 and 5 demonstrated some inhibitory activity. Thus, electrochemical redox reactions of CYP17A1 may serve as an adequate substitution of the reconstituted system, which requires additional redox partners for manifestation of catalytic activity of hemoproteins of the cytochrome P450 superfamily.
Keywords: recombinant form of human cytochrome P450 17A1; electrochemistry; inhibitors; pregnenolone

Some inhibitors of purine nucleoside phosphorylase by L. H. Pogosian; L. S. Nersesova; M. G. Gazariants; Z. S. Mkrtchian; J. I. Akopian (60-64).
Purine nucleoside phosphorylase (PNP) catalyzes reversible phosphorolysis of purine deoxy- and ribonucleosides with formation (d)Rib-1-P and corresponding bases. PNP plays a leading role in the cell metabolism of nucleosides and nucleotides, as well as in maintaining the immune status of an organism. The major aim of the majority of studies on the PNP is the detection of highly effective inhibitors of this enzyme, derivatives of purine nucleosides used in medicine as immunosuppressors, which are essential for creating selective T-cell immunodeficiency in a human body for organ and tissue transplantation. The present work is devoted to the study of the effects of some synthetic derivatives of purine nucleosides on activity of highly purified PNP from rabbit spleen and also from human healthy and tumor tissues of lung and kidneys. Purine nucleoside analogues modified at various positions of both the heterocyclic base and carbohydrate residues have been investigated. Several compounds, including 8-mercapto-acyclovir, 8-bromo-9-(3,4-hydroxybutyl)guanine, which demonstrated potent PNP inhibition, could be offered for subsequent study as immunosuppressors during organ and tissue transplantation.
Keywords: purine nucleoside phosphorylase; activity; substrates; inhibitors; tumor tissues; drug preparations

The cultivation of bone marrow cells and various cell lines on polymer films by M. S. Dolgikh; D. N. Livak; M. E. Krasheninnikov; N. A. Onishchenko (65-71).
The cultivation of multipotent mesenchymal stromal bone marrow cells (BM-MSC) and A-431, MDCK, Vero, 3T3 and Hep-G2 lines of cells was performed in DMEM medium using polyvinyl alcohol (PVA) polymer films as scaffolds. PVA was modified by covalent linking of various hydrophobic fatty acids. Different cell types exhibited different growth intensity on these films, which, however, in most cases was comparable with control cultivations on plastics.
Keywords: substrates; cultivation; polyvynil alcohol (PVA); bone marrow cells; cell lines

Enzyme-linked immunosorbent assay for determination of cyclosporin a in the whole blood by I. P. Andreeva; N. T. Vorobyeva; L. I. Vinnitsky; S. S. Bogush; E. M. Gavrilova; A. M. Egorov (72-75).
A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range of 60–1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radioimmunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.984, respectively. The developed test system is stable for at least 9 months when stored at 4°C and can be used in clinical practice.
Keywords: cyclosporin A; enzyme-linked immunosorbent assay (ELISA)

The state of the antioxidant system during therapy of patients with multiple sclerosis by L. P. Smirnova; N. V. Krotenko; E. V. Grishko; N. M. Krotenko; V. M. Alifirova; S. A. Ivanova (76-80).
Activity of erythrocyte glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GT), glucose-6-phosphate dehydrogenase (G6PDH), catalase and superoxide dismutase (SOD), the level of erythrocyte malonic dialdehyde (MDA) and also total antioxidant activity of blood serum were studied in patients with different types of multiple sclerosis (MS). Investigation of peripherical blood was carried out on the first day of admission to the hospital and after the standard therapy with copaxone. During the whole period of observation all MS patients had a high level of MDA and activity of erythrocyte GP compared with a control group. Other erythrocyte antioxidant enzymes and total antioxidant activity of blood serum exhibited weak positive dynamics in patients with relapsing-remitting multiple sclerosis (RRMS). The pathological decrease of antioxidant system activity in patients with secondary progressive multiple sclerosis (SPMS) was more pronounced and remained unchanged after the treatment. This is consistent with a more severe clinical course of this disease.
Keywords: oxidative stress; antioxidant enzymes; multiple sclerosis

Changes in the fatty acid composition of blood cell membranes in children with inflammatory diseases by N. M. Shilina; M. I. Dubrovskaya; O. N. Komarova; F. A. Medvedev; I. Ya. Kon (81-87).
Fatty acid composition of erythrocyte and leukocyte membranes has been studied in children (with normal body mass and obesity) with inflammatory diseases of the gastrointestinal tract and children with bronchial asthma in comparison with basically healthy children. Fifty seven children aged from 7 to 14 years were examined: 13 with inflammatory diseases of the gastrointestinal tract (IDGIT) (eosophagitis, gastroduodenitis, stomach ulcer), 25 with obesity stages (I–III) complicated by IDGIT, 9 with bronchial asthma and 10 basically healthy children. The study revealed that both IDGIT and bronchial asthma caused significant and similar changes in fatty acid composition of cell membranes. These included accumulation of ω3 eicosapentaenoic acid (EPA) and the decrease of docosahexaenoic acid (DHA); this phenomenon observed in both erythrocyte and leukocyte membranes suggests a common feature of the detected changes in fatty acid composition of cell membranes in inflammation. There was a significant decrease in the level of membrane ω6 polyunsaturated fatty acids (PUFA), first of all arachidonic acid and total ω6 PUFA. Consequently, EPA accumulation in membranes may be a compensatory response to low dietary arachidonic acid supply and/or its increased loss for the synthesis of pro-inflammatory eicosanoids (prostaglandins, leukotriens, thromboxans) during inflammatory process.
Keywords: inflammation; children; polyunsaturated fatty acids; blood cells; membranes

The proportion of mutant extracellular mitochondrial DNA increases in lung cancer patients after radiotherapy by I. Yu. Strelkova; S. A. Abdullaev; G. P. Snigireva; V. G. Bezlepkin; A. I. Gaziev (88-93).
Quantitative and qualitative changes in circulating extracellular DNA (ec-DNA) of blood plasma are considered as markers for diagnosis and prognosis of tumor pathology. We have investigated the content of mutant copies of the circulating mitochondrial DNA (ec-mtDNA) in blood plasma in 8 patients with lung cancer before and after radiotherapy as well as in healthy young and elderly donors. It was found that in plasma of healthy elderly donors the proportion of ec-mtDNA with mutations (in the total circulating DNA) is much higher than in young donors. Before radiotherapy the proportion of ec-mtDNA with mutations was higher in plasma of lung cancer patients (aged 70–76 years) than that of healthy elderly donors. After radiotherapy of lung cancer patients a twofold increase in the proportion of ec-mtDNA with mutations was observed in total circulating plasma DNA. This may be attributed to release of mutant DNA copies from dying tumor cells and also from normal cells injured by the radiation treatment.
Keywords: lung cancer; plasma extracellular mtDNA; radiotherapy; mutations; CEL-1 endonuclease; cleavage of heteroduplexes