Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.6, #4)

The influence of ambroxol (a mucolytic agent) on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside (SNP) and Sin-1) were investigated. Ambroxol in the range of concentrations from 0.1 to 10 μM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the SNP-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values of 3.9 and 2.1 μM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX.The influence of artemisinin (an antimalarial agent) on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1−100 μM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the SNP-induced activation of human platelet guanylate cyclase with the IC50 value of 5.6 μM. Artemisinin (10 μM) also inhibited (by 71 ± 4.0%) the activation of the enzyme by a thiol-dependent NO-donor, the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 μM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the signaling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.
Keywords: soluble guanylate cyclase; nitric oxide (NO); ambroxol; artemisinin

Mass spectrometry detection of monomeric renalase in human urine by V. I. Fedchenko; O. A. Buneeva; A. T. Kopylov; A. A. Kaloshin; L. N. Axenova; V. G. Zgoda; A. E. Medvedev (300-306).
Renalase is a recently discovered secretory protein, which is suggested to play a role (which still remains elusive) in regulation of blood pressure. Earlier it was purified from urine of healthy volunteers by means of ammonium sulfate fractionation and subsequent affinity chromatography (Xu et al. (2005), J. Clin. Invest., 115, 1275). The resultant purified preparation of renalase contained 2 proteins with molecular masses of 35 and 67–75 kDa. The authors believed that the latter represents a dimerization (aggregation) product of the 35 kDa protein. In this study we have detected relanase in urinary samples of 2 of 6 volunteers only after immunoaffinity enrichment of urinary samples subjected to ammonium sulfate precipitation. Electrophoresis of the purified preparation also demonstrated the presence of 2 proteins with molecular masses of 35 and 66 kDa, respectively. Mass spectrometry analysis of these proteins identified 35 and 66 kDa proteins as renalase and serum albumin, respectively. Thus, our results do not support suggestion on formation of renalase dimers and they indicate that urinary renalase excretion significantly varies in humans.
Keywords: renalase; urine; fractionation; immunoaffinity enrichment; mass spectrometry analysis

Antitumor activity of L-asparaginase from Erwinia carotovora against different human and animal leukemic and solid tumor cell lines by O. Yu. Abakumova; O. V. Podobed; P. A. Karalkin; L. I. Kondakova; N. N. Sokolov (307-316).
The dose- and time-dependent antitumor and cytotoxic effects of L-asparaginases from Erwinia carotovora (ECAR LANS) and Escherichia coli (MEDAC) have been investigated using human leukemic cells and human and animal solid tumor cells. These included human T-cell acute lymphoblastic leukemia cell lines (Jurkat, Jurkat/A4, Molt-4), human chronic myeloid leukemia K562 cells, human promyelocytic leukemia HL-60, and also human solid tumor cells (prostate carcinoma LnCap, breast adenocarcinoma MCF7, ovarian adenocarcinoma SCOV-3 and carcinoma CaOV, hepatocarcinoma Hep G2, fibrosarcoma HT-1080) and animal solid tumor cells (rat Gasser’s ganglion neurinoma cells GGNC-1, mouse glioblastoma EPNT-5). We investigated sensitivity of tumor cells (seeded at different density) to L-asparaginases, as well the effect of L-asparaginases on cell growth rate, protein and DNA synthesis in the presence of various cytostatics. Cell cycle analysis by flow cytofluorimetry and detection of apoptotic cells before and after treatment with L-asparaginases indicate that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division. The cytofluorometric study of solid and leukemic cells revealed that except HL-60 cells the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. Combined treatment of cells using a combination of L-asparaginase and doxorubicin significantly increased the number of apoptotic cells up to 60% (MCF-7 cells), 40% (Jurkat cells) and even 99% (HL-60). High levels of DNA and protein synthesis rates in asparaginase-treated tumor cells suggest lack of massive entry of tumor cells to apoptosis. This conclusion is based on the fact of sensitivity of multi-resistant Jurkat/A4 cells to L-asparaginases (it is nearly impossible to induce apoptosis in these cells). Since ECAR LANS did not influence growth of normal human fibroblasts it appears that the enzyme cytotoxicity is associated only asparagine deficiency.
Keywords: L-asparaginase; tumor cells; cytotoxicity; DNA and protein synthesis; apoptosis; Erwinia carotovora

Binase penetration into alveolar epithelial cells does not induce cell death by H. A. Cabrera-Fuentes; N. V. Kalacheva; R. T. Mukhametshina; P. V. Zelenikhin; A. I. Kolpakov; G. Barreto; K. T. Preissner; O. N. Ilinskaya (317-321).
Microbial ribonucleases possess a broad spectrum of biological activities, which demonstrate stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. Mechanisms of their penetration into the cells still remain unclear. In this study penetration of Bacillus intermedius RNase (binase) in alveolar lung epithelial cells, type II (ATII) pneumocytes, has been investigated. Using immunofluorescence analysis we have shown for the first time internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in MLE-12 (Murine Lung Epithelial-12 cells). However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. These results clearly indicate higher sensitivity of tumor cells to binase compared to normal cells; they also demonstrate that penetration of the enzyme into alveolar epithelial cells is not directly associated with their death.
Keywords: binase; cytotoxicity; internalization; immunofluorescence; type II alveolar epithelial cells; MLE-12; ATII

The activity of liver microsomal and Guerin’s carcinoma NADH-cytochrome b 5 reductase, the content and the rate of cytochrome b 5 oxidation-reduction have been investigated in tumor-bearing rats exposed to preliminary irradiation. Preliminary irradiation of rats (before transplantation of Guerin’s carcinoma) resulted in the decrease of NADH-cytochrome b 5 reductase activity and the content of cytochrome b 5 in the Guerin’s carcinoma microsomal fraction in the latent and logarithmic phases of oncogenesis compared with the non-irradiated tumor-bearing rats. The effect of irradiation preceding transplantation of the tumor to rats results in the increase of enzymatic activities of liver microsomal NADH-cytochrome b 5 reductase in the latent and logarithmic phases of tumor growth as compared with non-irradiated tumor-bearing rats. At the same time the content of cytochrome b 5 decreased, while the rate of its oxidation-reduction rate simultaneously increased in the liver microsomal fraction of tumor-bearing rats.
Keywords: NADH-cytochrome b 5 reductase; cytochrome b 5 ; microsomal fraction; liver; Guerin’s carcinoma

Administration of a synthetic compound with predicted anti-ischemic and cardioprotective activity, 3,5-dicarbomethoxyphenylbiguanide (3,5-DCMPBG) to rats with experimental myocardial infarction led to a decrease in the lipid peroxidation level, glutathione peroxidase activity, the level of reduced glutathione, activity of NADP-isocitrate dehydrogenase in the heart and blood serum, and activity of glucoso-6-phosphate dehydrogenase in heart in comparison with their levels in untreated animals with myocardial infarction. This may be attributed to a decrease of free radical processes and reduction of antioxidant system loading induced by the protective effect of the administered compound. At the same time the increase glutathione reductase activity observed under these conditions in the heart and blood serum is probably associated with specific influence of 3,5-DCMPBG on this enzyme.
Keywords: experimental myocardial infarction; glutathione antioxidant system; 3,5-dicarbomethoxyphenyl-biguanide

Hypoxic preconditioning modifies activity of pro- and antioxidant systems in the rat hippocampus by M. S. Kislin; S. A. Stroev; T. S. Gluschenko; E. I. Tyulkova; M. Pelto-Huikko; M. O. Samoilov (333-337).
The effects of repetitive mild hypobaric hypoxic preconditioning on pro- and antioxidant systems in rat hippocampus have been studied. It was found that three-trial preconditioning by mild hypobaric hypoxia (360 mm Hg, 2 h) induced moderate oxidative stress immediately after the last preconditioning trial. In addition, it down regulated the levels of protein antioxidants (Trx-1, Trx-2, Cu,Zn-SOD) and also decreased several lipid peroxidation products 24 h after the preconditioning.
Keywords: preconditioning; hypobaric hypoxia; antioxidants; lipid peroxidation; hippocampus

Effect of exogenous antioxidants on erythrocyte redox status and hepcidin content in disorders of iron metabolism regulation by S. P. Scherbinina; A. A. Levina; I. L. Lisovskaya; F. I. Ataullakhanov (338-342).
In many diseases associated with impairments in iron metabolism, erythrocytes exhibit an increased sensitivity to oxidative stress induced in vitro. In this study we have examined the antioxidant status of erythrocytes from healthy donors and from 12 patients with disorders of iron homeostasis by measuring the extent of hemolysis induced in vitro by tert-butyl hydroperoxide (t-BHP). The extent of hemolysis observed with patient erythrocytes was significantly higher than that observed in experiments with erythrocytes from healthy donors. After therapeutic infusions of the antioxidants mexidol or emoxypin, oxidative hemolysis in patients was restored to normal values and blood hepcidin increased significantly as compared with its initial level. A significant correlation was observed between hepcidin concentration after treatment and t-BHP-induced hemolysis before treatment. These data suggest that antioxidants may exert a favorable effect on those at risk for iron overload disease.
Keywords: erythrocytes; iron metabolism; hepcidin; tert-butyl hydroperoxide; antioxidants

The review considers the current knowledge on molecular mechanisms of apoptosis. Particular emphasis is given to the key elements of the extrinsic death receptor pathway and the intrinsic mitochondrial pathway. Dysregulation of apoptotic pathways is considered as a key factor in the survival of cancer cells in response to conventional chemotherapeutic drugs or radiation therapy. Substances that selectively reactivate apoptosis in malignant cells are considered as the promising candidate anticancer drugs, which have now entered various phases of clinical trials. The modern techniques allowing non-invasive visualization of apoptotic cells with special reference to therapy-induced cell death are briefly surveyed.
Keywords: apoptosis; targeted therapy; cancer patients; clinical trials; single-photon emission computed tomography; positron emission tomography; extracellular markers of apoptosis