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

The great importance of glutathione as a redox regulator and the reducing carrier has been recognized. There is clear necessity for subdivision of an independent mitochondrial glutathione subsystem. The data on specific features of glutathione metabolism in different organs are accumulated. There is convincing evidence for the involvement of the glutathione system into inflammation and immunity. Studies of the glutathione system are used in medicine for elucidation of pathogenesis of diseases and their diagnostics.
Keywords: glutathione; metabolism enzymes; thiol-disulfide; inflammation; immunity; functions

Stem cells of intestinal epithelium. The mechanisms of survival and the role of microbiota by S. Ya. Proskuryakov; A. G. Konoplyannikov; L. P. Ulyanova; D. Yu. Logunov; B. S. Narodicky; A. L. Gincburg (221-236).
The thanatogenetic mechanisms of stem cells (sc) of rapidly renewing system of intestinal epithelium still remain unclear. on the one hand they are definitely involved into basic mechanisms of carcinogenesis in the gastrointestinal tract, because dysregulation of programs responsible for elimination of “unwanted” mutant cells (which are normally under immune and own intrinsic control) is one of the reasons of neoplastic expansion. on the other hand elucidation and characterization of the regulatory machinery controlling sc survival are interrelated with problems of clinical medicine, including the increase of therapeutic efficiency of treatment of inflammatory and ulcer lesions of the gut, traumatic and surgical wounds, as well as restriction of side effects in normal tissues induced by application of intensive methods chemo- and radiotherapy of cancer. the latter is especially important for treatment of blood diseases and tumors of peritoneal cavity organs mainly due to bone marrow and intestinal epithelium damage. (These tissues are the most sensitive to these treatments.) The review considers data on exogenous and genetic modifiers of sc survival, and also the basic principles of mechanisms involved into renewal and regeneration of sc and the effects of microbiota on these processes.
Keywords: intestinal epithelium; stem cells; crypt clonogenic cells; modifiers of destruction/survival rate; Wnt-β-catenin signaling pathway

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), exhibiting different biochemical properties. The catalytically active ACE isoforms consisted of just one domain have been also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when their crystal structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control of different physiological functions. The C-terminal catalytic domain plays an important role in the regulation of blood pressure: it catalyzes angiotensin I cleavage in vivo. The N-domain contributes to the processing of other bioactive peptides for which it exhibits high affinity. The role of the N-domain is not ultimately associated with functioning of the rennin-angiotensin system and it contributes processing of other bioactive peptides for which it exhibits high affinity (goralatide, luliberin, enkephalin heptapeptide, beta-amyloid peptide). Domain-selective inhibitors selectively blocking either the N- or C-domain of ACE have been developed.
Keywords: angiotensin converting enzyme; domains; crystal structure; domain-specific substrates; domain-selective inhibitors

Human embryonic stem cells (hESC) are able to maintain pluripotency in culture, to proliferate indefinitely and to differentiate into all somatic cell types. Due to these unique properties, hESC may become an exceptional source of tissues for transplantation and have a great potential for the therapy of incurable diseases. Here, we review new developments in the area of embryonic stem cells and discuss major challenges — standardization of protocols for cell derivation and cultivation, identification of specific molecular markers, development of new approaches for directed differentiation, etc. — which remain to be settled, prior to safe and successful clinical application of stem cells. We appraise several potential approaches in hESC-based therapy including derivation of autologous cells via therapeutic cloning (1), generation of immune tolerance to allogenic donor cells via hematopoetic chimerism (2), and development of the banks of hESC lines compatible with the main antigens and exhibiting equivalent pluripotency (3). In addition, we discuss briefly induced pluripotent cells, which are derived via genetic modification of autologous somatic cells and are analogous to ESC. Our analysis demonstrates that uncontrollable differentiation in vivo and teratogenic potential of hESC are critical limitations of their application in clinical practice. Therefore, the major approach in hESC therapy is derivation of a specific differentiated progeny, which has lower proliferative potential and immune privilege, yet poses fewer risks for organism. The review demonstrates that cell therapy is far more complex and resource-consuming process as compared with drug-based medicine and consequently pluripotent stem cell biology and technology still requires further investigation and development before these cells can be used in clinical practice.
Keywords: embryonic stem cells; induced pluripotent cells; cell therapy

Cystatins are thiol proteinase inhibitors ubiquitously present in the mammalian body. In brain, they prevent unwanted proteolysis and are involved in several neurodegenerative diseases. Under physiological conditions nitric oxide can be found in almost all the tissues, but under pathological conditions NO has damaging effects. Its increased concentration, under various neural diseases leads to cell damage through formation of highly reactive peroxynitrite. Our present study was designed to investigate the protective effect of curcumin against NO induced damage of HM-GBC. NO caused intensive structural and functional damage of HM-GBC, resulting in 89% loss of its antiproteolytic activity after 2 h of incubation. Structural damage occurs in the form of protein degradation. Curcumin significantly protected HM-GBC against this damage. This suggests that curcumin has a significant potential in the treatment of diseases caused by nitrogen free radicals and this potential must be further explored for the development of novel drugs.
Keywords: nitric oxide; high molecular mass goat brain cystatin; antiproteolytic activity; curcumin

Expression of gelatinases A and B and their endogenous regulators in immortal and transformed fibroblasts by N. I. Solovyeva; S. V. Vinokurova; O. S. Ryzhakova; T. A. Gureeva; I. V. Tsvetkova (266-271).
Matrix metalloproteinases (MMP) play a critical role in tumor invasion and metastasis. The goal of this study was to elucidate peculiarities of expression of gelatinases A and B (MMP-2 and MMP-9), membrane type MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP-2) in immortal (IF) and transformed fibroblasts (TF). The study was carried out using embryo rat fibroblasts, sequentially immortalized with the polyomavirus LT gene and transformed with the E7 gene of human papillomavirus (HPV-16). Papillomaviruses type16 and 18 are the etiological factor for cervical cancer. A primary fibroblast (PF) culture of Fisher rats was used as control. Analysis of TF and IF included determination of MMP-2 and MMP-9 activity by hydrolysis of the specific substrate, radioactive collagen type IV; analysis of MMP spectra by a zymographic assay, and estimation of the mRNA expression by RT-PCR. It was found that: (1) collagenolytic activity of MMP was increased only in TF and it depended on the degree of cell tumorigenicity; (2) the study of MMP spectra revealed the presence of MMP-9 only in TF, whereas MMP-2 was found in IF as well; (3) the mRNA expression of MMP-9, MT1-MMP and TIMP-2 increased in all TF while the MMP-2 expression increased in TF only after TF cell selection on rats; (4) the collagenolytic activity as well as the mRNA expression of MMP-2 and MMP-9 and endogenous regulators (MT1-MMP and TIMP-2) did not change in immortalized fibroblasts compared to the PF culture. The data obtained indicate changes in the ratio enzyme/activator/inhibitor and also suggest a significant increase in the TF destructive potential. MMP-9 is supposed to be a marker of fibroblasts transformed by E7 HPV16 gene in a cell culture.
Keywords: gelatinases A and B; MMP-2; MMP-9; MT1-MMP; TIMP-2; immortalization; transformation; gene E7 HPV-16

Biosensor analysis of the interaction of potential dimerization inhibitors with HIV-1 protease by P. V. Ershov; O. V. Gnedenko; A. A. Molnar; A. V. Lisitsa; A. S. Ivanov; A. I. Archakov (272-288).
Inhibitors of protein-protein interactions are currently considered as perspective prototypes of a new generation of drugs. The most attractive targets for such inhibitors are the oligomeric enzymes which active sites are formed by amino acid residues from different subunits. HIV-1 protease (HIVp), which is active only as a homodimer form, is the classic example of such enzymes. We have developed a new approach for experimental screening of HIVp dimerization inhibitors. It is based on an original biosensor test-system for differential analysis of interaction of tested substances with HIVp dimers and monomers. Using this test-system we have analyzed the most perspective candidate substances predicted by the method of virtual screening, and also some derivatives of glycyrrhizin, triterpenic and steroid glycosides. In the results of this study we have found one compound, which preferentially interacts with HIVp monomers and inhibits in vitro activity of this enzyme with the IC50 value of about 10−6 M.
Keywords: HIV-1 protease; dimerization; dimerization inhibitors; validation; optical biosensor; surface plasmon resonance

The effect of myosin ATPase inhibitor, 2,3-butanedione monoxime (BDM) used in the range of concentrations 1.25–10.0 mM), on recovery of functions of isolated rat heart subjected to normothermic (37 °C) total ischemia for 35 min has been investigated. BDM perfusion was performed at a flow rate of 4 ml/min during 5 min before ischemia (BDM-I) or before 25-min reperfusion (BDM-R). Control hearts were perfused with Krebs solution at the same flow rate. The highest functional recovery of heart and coronary vessels was observed during infusion of 2.5 mM BDM before ischemia. At the end of reperfusion ATP and phosphocreatine (PCr) content in hearts of this group was significantly higher whereas the level of lactate was two times lower than in control; total creatine content (ΣCr) did not differ from the initial level. Similar but less pronounced changes in the improvement of aerobic metabolism and maintenance of ΣCr after reperfusion were also observed in the case of infusion of 2.5 mM BDM before reperfusion. They were consistent with reduced recovery of functions of heart and coronary flow compared with these parameters observed in the BDM-I group. 2.5 mM BDM caused almost 2-fold decrease in release of cardiac lactate dehydrogenase into myocardial perfusate in the BDM-I and BDM-R groups (compared with control); this suggests lower damage of cell membranes. These results suggest that improvement of energy supply of postischemic cardiomyocytes may be a key factor determining cardioprotector effectiveness of short-term administration of BDM before ischemia.
Keywords: ischemia and reperfusion; 2,3-butanedione monoxime; macroergic phosphates; lactate; cardiomyocyte membranes; function of heart and coronary vessels

Lysosomal cathepsins B, L, and D in the development of murine experimental leukemias by T. A. Khalikova; T. A. Korolenko; S. I. Ilnitskaya (295-303).
Lysosomal proteases are actively involved into pathogenesis of malignant tumors. Impairments in the interaction between proteases and their inhibitors are implicated in the processes of tumor invasion and metastasis. Among proteases associated with malignant growth, cysteine cathepsins B and L and aspartic cathepsin D are considered to play the major role in the tumor development. The present study was designed to investigate the activity of cathepsins B, L, and D during the development and treatment of murine experimental leukemias and to determine correlation between these proteases and course of pathological process as well as efficiency of the chemotherapeutic treatment. P-388 leukemia was characterized by a more aggressive development and unfavorable prognosis than L1210/1 leukemia. In mice with P-388 leukemia the activity of lysosomal cathepsins B, D, and L in the tumor tissue, liver and spleen, as well as the activity of cathepsins B and L in serum were lower than activities of these enzymes in mice with L1210/1 leukemia. Changes in the activity of cathepsins in liver and spleen of leukemic mice reflected a level of aggressiveness of the tumor development and invasion of these organs with tumor cells. Treatment of these experimental leukemias resulted in the increase of cathepsin B, L and D activity in the tumor tissue, liver, spleen and the increase in cathepsin B and L activity in serum. The highest protease activity was detected in the groups of mice characterized by the highest inhibition of the tumor growth. These data demonstrate that lysosomal proteases are involved in the progression of murine experimental leukemias and elimination of tumor cells in the result of treatment. Thus, determination of the activity of cysteine and aspartic proteases can be used for evaluation of cancer malignancy, tumor sensitivity for chemotherapy and efficiency of treatment.
Keywords: cysteine proteases; aspartic proteases; transplanted leukemias; cyclophosphamide

A new method for quantitative estimation of the virus particles number by A. S. Shebanova; M. N. Savvateev; N. V. Maluchenko; D. Yu. Trofimov; I. I. Agapov (304-310).
The virus particles of live mumps virus vaccine widely used for vaccination in Russia have been detected and visualized by the atomic force microscopy. For quantitative estimation of the number of observed virus particles the special method has been developed. The presence of the vaccine virus protein component was tested by ELISA and dot-blot analysis. Using a quantitative real-time PCR assay the number of copies of viral RNA was estimated. The results of the quantitative estimation obtained by real-time PCR corresponded to the atomic force microscopy data.
Keywords: atomic force microscopy; live virus vaccine; virus particles; quantitative real-time PCR

The regulatory effects of insulin, insulin-like growth factor 1 (IGF-1), and relaxin on glucose-6-phosphate dehydrogenase (G6PDH) and glycogen synthase (GS) activities have been studied in myometrium of pregnant women of control group and with diabetes mellitus of different etiology. In patients with type 1 diabetes G6PDH activity did not differ from the control group, but the enzyme activity was sharply decreased in pregnant women with type 2 diabetes and gestational diabetes. In the control group maximal stimulation of G6PDH activity was observed at 10−9 M of peptides and their stimulating effect decreased in the following order: insulin > relaxin > IGF-1. In pregnant women with types 1 diabetes insulin effect on the enzyme activity was lower than in the control, and the effects of IGF-1 and relaxin were absent. In the group of pregnant women with type 2 diabetes and gestational diabetes the effects of insulin and IGF-1 were decreased, but the effect of relaxin was somewhat higher thus giving the following order in their efficiency relaxin > IGF-1 = insulin. At 10−9 M peptides exhibited similar stimulating effects on the active form of GS-I, but had no influence on the total enzyme activity in the control group of pregnant women. In patients with type 1 diabetes GS activity remained unchanged (versus control), and peptides did not stimulate the enzyme activity. In patients with type 2 diabetes a significant decrease in GS activity was accompanied by the decrease in the effect of peptides, giving the following order of their efficiency: insulin = IGF-1 > relaxin. In myometrium of pregnant women with gestational (treated and untreated) diabetes GS activity decreased, the effect of insulin was weaker, whereas the effects of relaxin and IGF-1 increased thus giving the following order of their efficiency: relaxin > IGF-1 > insulin. Insulin therapy of type 1 diabetes incompletely restored sensitivity of the enzymes to the peptide actions. At the same time, in women with gestational diabetes and subjected to insulin therapy the stimulating effect of relaxin on the enzyme activities increased. This fact suggests that relaxin exhibits replacement functions under conditions of attenuated insulin action.
Keywords: insulin; glucose-6-phosphate dehydrogenase; glycogen synthase; myometrium; pregnancy; diabetes

Placental enzymes involved in regulatory peptide metabolism in EPH-gestosis by O. P. Petrushova; M. T. Gengin; V. A. Smetanin; A. V. Kuznetsova (317-321).
Essential edema-proteinuria-hypertension (EPH) gestosis still represents an important obstetrical problem. We have investigated the activity of carboxypeptidase H (CPH), phenylmethylsulfonyl fluoride inhibited carboxypeptidase (PMSF-CP), carboxypeptidase M (CPM) and angiotensin-converting enzyme (ACE), the main carboxypeptidases in human placenta under normal conditions and mild EPH-gestosis. Gestosis was accompanied by the decrease in activity of the enzymes involved into metabolism of regulatory peptides (ACE, CPH, PMSF-CP, CPM) compared with their activity in placenta under physiological pregnancy. Correlation analysis revealed positive correlation between placental CPH and CPM (r = 0.2735*) in EPH-gestosis. These findings suggest involvement of placental proteases into formation of compensatory-adaptive reactions in the fetoplacental complex at EPH-gestosis; the data obtained may be also employed for the development of methods of prophylaxis and corrections of metabolic impairments in pathology of pregnancy.
Keywords: placenta; fetoplacental complex; EPH-gestosis; carboxypeptidase; angiotensin-converting enzyme