Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.2, #3)

Steroids, histamine, and serotonin in the medicinal leech salivary gland secretion by I. P. Baskova; Z. Ferner; A. S. Balkina; S. A. Kozin; O. V. Kharitonova; L. L. Zavalova; V. G. Zgoda (215-225).
Lipids represent 20% of the total weight of the dried pool of medicinal leech salivary gland secretion (SGS) obtained from about 50 individual animals. SGS lacks phospholipids, but contains steroids. Immunochemiluminescent analysis of SGS revealed the presence of free steroid hormones: cortisol, progesterone, testosterone, estradiol, and dehydroepiandrosterone. Micro-chromatographic-mass spectrometric analysis of SGS and its low molecular weight fraction (LMW) (molecular masses ranged from 220 to 850 Da) has shown the multicomponent nature of the LMW fraction. Using standard preparations as the reference steroid hormones (cortisol, dehydroepiandrosterone, androstenedione, and testosterone) and histamine and serotonine have been identified in SGS.
Keywords: lipids; steroid hormones; histamine; serotonin; salivary gland secretion; medicinal leech

Comparative transcriptomics and proteomics of fetal and adult human liver by L. K. Kurbatov; I. B. Cheglakov; K. N. Yarygin; G. T. Sukhikh; G. V. Vartanian; I. Yu. Toropygin; A. I. Archakov (226-234).
The comparative transcriptome and proteome analysis of the fetal liver (10.5 weeks of gestation) and adult liver was carried out. Data on differential gene expression were obtained using two-color fluorescent probe competitive hybridization of fluorescently labeled cRNA and Agilent’s whole human genome microarrays. Protein profiles were obtained by means of two dimensional gel electrophoresis (2D-GE) of cell extracts within the range of pI from 3 to 10. Proteins isolated from gels were identified by MALDI-TOF mass spectrometry. The list of identified proteins was then compared with the list of genes preferentially expressed in fetal liver. The research object, fetal liver, is a unique organ because it combines both hepatic and hematopoietic tissues and during embryogenesis liver changes its main functional activity from hematopoietic (at early stages of development) towards metabolic functions (including detoxification and secretion of various serum proteins). Data obtained show that transcriptome analysis adequately reflects the main hepatic function during early stages of embryogenesis. Hemoglobins of both embryonic and adult types were identified. Results of proteomic analysis were basically confirmed during mRNA analysis, however, some protein products of highly expressed genes were not detected during 2D-GE.
Keywords: two-dimensional electrophoresis; expression analysis; DNA microarrays; gene expression; fetal liver

The Biopharmaceutical Classification System (BCS) is based on solubility tests, correlating for certain drugs with their bioavailability in human body. It is widely used in design and development of innovation drugs, new dosage forms (permeability amplifiers), in clinical pharmacology (drug-drug, drug-food interaction) and also by regulation agencies of several countries as the scientific approach, for testing of waiver on bioavailability. The review considers modern concepts and theoretical bases for prediction of bioavailability according to BCS. It gives characteristics of fundamental parameters of the system: absorption number, solubility number and ratio of dose to the soluble part of the drug. Possible versions of BCS modification for its subsequent optimization are discussed.
Keywords: biopharmaceutical classification system (BCS); bioavailability; generic drug

The review characterizes the main enzymatic systems of selective proteolysis responsible for maintenance of intracellular proteome in prokaryotes, eukaryotes and archaea. The features of proteolytic components of the ATP-dependent proteases as well as similarity and diversity of their regulatory components belonging to AAA+ ATPases are discussed.
Keywords: AAA+ proteins; ATP-dependent proteases; selective proteolysis; proteasome; prokaryotes; eukaryotes; archaea

Putative hepatitis C virus cell receptors by T. E. Farafonova; L. V. Olenina; E. F. Kolesanova (258-266).
Virus entry into a host cell comprises the first step of the viral life cycle. Blockage of this process can stop or prevent the rise of the infection. Development of compounds exhibiting directed blocking activity requires information about host cell and viral molecules, which are involved into reciprocal recognition resulting in the virus entry into the cell. This review is devoted to the problems of the identification of plasma membrane molecules, involved in binding of hepatitis C virus and its subsequent transfer inside the cells. The putative role of these molecules as hepatitis C virus receptors and co-receptors in the beginning and development of the infection is discussed.
Keywords: hepatitis C virus; receptors; envelope glycoproteins

The role of carboxypeptidases in carcinogenesis by I. L. Vovchuk; S. A. Petrov (267-274).
The literature and own experimental data on the role of metallocarboxypeptidases in carcinogenesis have been reviewed. The development of various tumors is accompanied by the increase in activity of all groups of these. It is suggested that in some cases carboxypeptidases play a protective role attributed to inhibition of tumor development.
Keywords: carboxypeptidases; carcinogenesis

The insufficiency of liver functions remains one of the major causes of death. The liver transplantation is the most effective method for treating severe liver diseases. The shortage of donor organs and high risk of graft rejection are the main problems for liver transplantation. Stem cells and isolated hepatocytes are the alternative means for repopulating liver after various injuries instead of liver transplantation. This review analyses achievements in therapy of liver insufficiency by means of stem cells in model experiments on animals as well as in clinical practice and also perspectives of employment of stem cells for treatment of liver insufficiency.
Keywords: cell therapy; stem cells; liver insufficiency

The effect of ozonated saline on proteolytic enzymes (trypsin, chymotrypsin, elastase, kallikrein, leucine aminopeptidase), inhibitors of proteolysis and lipid peroxidation (LPO) has been investigated. Injection of ozonated saline caused marked response of the proteolytic system. Low ozone doses did not cause activation of proteolytic enzymes, whereas high doses activated proteases, decreased the level of inhibitors of proteolysis (α1-antitrypsin and α2-macroglobulin) and stimulated accumulation of LPO products. Thus, analyses of proteolytic activity can be used as an indicator of effectiveness and safety of ozone therapy and other treatment programs.
Keywords: ozone; trypsin; kallikrein; elastasa; pancreas

Synthesis of the β-amyloid fragment 5RHDSGY10 and its isomers by E. Yu. Aleshina; N. V. Pyndyk; A. A. Moisa; M. A. Sanzhakov; O. N. Kharybin; E. N. Nikolaev; E. F. Kolesanova (288-292).
The peptide RHDSGY, a fragment of the human β-amyloid Zn-binding site, and its isomers RH(D-Asp)SGY and RH(β-Asp)SGY have been obtained as amides by means of solid-phase synthesis and analyzed by HPLC and various mass spectrometric methods. The problem of low yield of the RHDSGY peptide and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of such side-products as RH(β-Asp)SGY (or RHDSGY during synthesis of RH(β-Asp)SGY) and RH(Asp-imide) SGY was solved via selection of individual reagents for removal of Fmoc groups from α-amino groups of the growing peptide chain.
Keywords: β-amyloid; solid-phase peptide synthesis; β-Asp; Fmoc-group removal

Cystatins: Cysteine proteases regulation and impairments in tumors and inflammation diseases by T. A. Korolenko; T. G. Filatova; M. S. Cherkanova; T. A. Khalikova; I. Yu. Bravve (293-297).
Cystatin C belongs to the most widespread group of endogenous extracellular cysteine protease inhibitors; its biological functions are investigated now. Using the ELISA method we have demonstrated that the highest cystatin C concentration is registered in human cerebrospinal fluid; significantly lower cystatin C lever is detected in human urine. In healthy individuals of young age serum cystatin C concentration is lower than in elder persons (of 50–65 years old). In patients with hemoblastoses (lymphoma, lymphogranulomatosis) increased serum cystatin C concentration was normalized after effective antitumor therapy. This suggests that serum cystatin C concentration can be used as one of the prognostic criteria in patients with several types of hemoblastoses.
Keywords: cystatins; cysteine proteases; tumors; atherosclerosis

Matrix metalloproteinases 2 and 9 as the factors of head and neck tumor metastases by I. V. Kondakova; E. V. Klisho; O. V. Savenkova; G. V. Kakurina; E. L. Choinzonov; D. A. Shishkin; M. R. Mukhamedov (298-301).
The levels of metalloproteinases (MMP-2,-9), their tissue inhibitors (TIMP-1,-2) and extracellular matrix metalloproteinase inducer (EMMPRIN) were studied in tumor tissue and blood serum from patients with head and neck squamous cell carcinoma. Immunohistochemical investigation showed much higher expression of MMP-9 and TIMP-1 in tumor tissue compared with MMP-2 and TIMP-2. There was different distribution of the investigated parameters (except TIMP-1) in cancer cells and stroma. Accumulation of MMP-2, MMP-9, and TIMP-2 was found mainly in cell elements (fibrocytes, leukocytes, etc.) and in stromal extracellular space. Expression of EMMPRIN was significantly higher in tumor cells than in stromal cells. It is possible that carcinoma cells express EMMPRIN, which may increase MMP production by surrounding cells. There was significant decrease of TIMP-1 expression in carcinoma cells with N1 grade of metastasis than in tumors without metastasis. The level of TIMP-1 in blood serum from patients with tumor metastases to regional lymph nodes was lower than in serum from patients without metastases. Thus, MMP-9 and TIMP-1 play an important role in the development of head and neck squamous cell carcinoma and the TIMP-1 level in blood serum and cancer tissues is linked to the first grade of regional lymph node metastasis.
Keywords: matrix metalloproteinases; tissue inhibitors of metalloproteinases; extracellular matrix metalloproteinase inducer; head and neck squamous cell carcinoma; metastasis

The effect of melatonin on free radical homeostasis in rat tissues at thyrotoxicosis by S. S. Popov; A. N. Pashkov; T. N. Popova; V. I. Zoloedov; A. V. Semenikhina; T. I. Rakhmanova (302-305).
Experimental thyrotoxicosis in rats is accompanied by the increase of serum alanine aminotransferase (AlAT), aspartate aminotransferase (AsAT), creatine phosphokinase-MB (CPK-MB) activities and the content of primary products of lipid peroxidation, conjugated dienes, in liver, heart and blood. This suggests impairments in functioning of these organs, which accompany intensification of free radical processes. Melatonin administration resulted in the decrease of AlAT, AsAT, CPK-MB and conjugated dienes; this indicates positive effect of melatonin in this pathology. Thyrotoxicosis is accompanied by the increase of catalase activity in rat liver, heart and serum. Exogenous melatonin decreased specific activity of serum and heart catalase by 22 and 43%, respectively, compared with rats subjected to hyperthyroidism. However, there was insignificant increase in specific activity of liver catalase (by ∼15%). Melatonin administration caused a decrease of α-tocopherol content increased in rat tissues under conditions of hyperthyroidism. Thus, exogenous melatonin is capable to reduce intensity of lipid peroxidation in hyperthyroidism and to act as an adaptogen, regulating free radical homeostasis in response to action of pathogenic factors on organism that is associated by concomitant reduction of mobilization of components of the antioxidant system.
Keywords: thyrotoxicosis; melatonin; free radical homeostasis; catalase; α-tocopherol

The influence of alimentary microelementosis on the activity of superoxide dismutase and glutathione peroxidase by A. V. Vasil’ev; V. I. Ivakhnenko; S. A. Khotimchenko; V. V. Korzh (306-310).
Studies of Km for glutathione peroxidase (GPx) and activities of superoxide dismutase and GPx were carried out in liver and erythrocytes of rats kept on either the normal semisynthetic diet or a high-fat diet with increased content of Cu, Zn, Mn, and Se. The diet containing microelement additions caused the increase in TBH affinity of liver and erythrocyte GPx, as well as the decrease of liver SOD observed on the 14th day of the treatment of rats with the high-fat diet with additional increase of Cu, Zn, Mn, and Se.
Keywords: superoxide dismutase; glutathione peroxidase; Cu; Zn; Mn; Se

Antibiotic sensitivity has been investigated in nosocomial bacterial Klebsiella spp. strains isolated from patients treated in 30 hospitals of 15 Russian regions. Among Klebsiella strains (n = 212) studied the following species were found: Klebsiella pneumoniae ss. pneumoniae—182 (85.8%), Klebsiella pneumoniae ss. ozaenae—1 (0.5%), Klebsiella oxytoca—29 (13.7%) strains. Their sensitivity to antibacterial preparations was estimated by the method of serial dilutions in microvolume (the microdilution method). Carbapenems (imipenem and meropenem) exhibited the highest antibacterial activity against the strains studied. Among third generation cephalosporins the lowest MIC (Minimum inhibitory concentration) were found in the inhibitor protected preparations: ceftazidime/clavulanic acid (MIC50 of 0.25 μg/ml; MIC90 of 64 μg/ml) and cefoperazone/sulbactam (MIC50 of 16 μg/ml; MIC90 of 64 μg/ml). Using the PCR method the detection of class A betalactamases genes (TEM, SHV, CTX) was carried out in 42 strains of Klebsiella pneumoniae ss. pneumoniae. TEM type beta-lactamases were found alone or in various combinations in 16 (38.1%) strains, SHV—in 29 (69%), and CTX—in 27 (64.3%). Combinations of 2 and 3 different resistance determinants were detected in 23.8 and 26.2% of strains, respectively. Screening of carbapenem-resistant Klebsiella strains for production of class B metallo-beta-lactamases did not reveal nosocomial strains with phenotypically documented production of these enzymes.
Keywords: nosocomial infections; Klebsiella spp; antibiotic resistance; cephalosporins; beta-lactamases; metallo-beta-lactamases

In our early experimental studies (with transplanted W-256, SM-1 and PC-1 tumors) and clinical observations (breast, urinary bladder and prostate cancers) we have investigated the effect of the anticancer drug NSC-631570 (known as Ukrain®) as an effective agent, which is selectively accumulated in a tumor tissue (after a single dose intravenous administration) and controls cancer-induced metabolic imbalance. This drug inhibits metabolic processes in the tumor and causes metabolic disorders in cancer cells. In this study we have investigated the effect of NSC-631570 on a pool of free amino acids in patients with malignant tumors. Blood plasma was sampled from 10 healthy donors and 29 patients with various types of cancer (stomach, rectal, lung, breast, urinary bladder, prostate cancers, chronic lympholeucosis, non-Hodgkin lymphoma, myeloma disease). Comparison of amino acid concentrations in blood plasma of tumor patients and healthy donors has shown: (1) selective effect of NSC-631570 on the level of amino acids with charged (His, Arg) or uncharged (Tyr, Thr, Gln) R-groups; (2) the decrease of His concentration and the increase in β-Ala and Tau concentrations. These changes depend on added drug concentration and localization of primary tumor process. Based on literature data and obtained results it is suggested that one of mechanisms responsible for realization of the antitumor effects of NSC-631570 consists in selective interaction with components of this preparation with amino acids, their derivatives and blood plasma proteins.
Keywords: amino acids; anticancer effects; NSC-631570; cancer diagnostics