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

Monoclonal antibodies in diagnostics of high-grade gliomas by V. P. Baklaushev; K. A. Pavlov; V. P. Chekhonin (105-115).
The review considers the modern methods of radioimmune diagnostics of high-grade gliomas using monoclonal antibodies and also other approaches of multi-component radioimmunolocalization with pretargeted bispecific antibodies. High-grade tumor-related proteins have been analyzed as potential targets for radiolabeled antibodies. Recent experimental and clinical data on immunolocalization of brain tumors and the most promising immunochemical approaches for diagnostic and targeted therapy of gliomas with tumor-specific antibodies are discussed.
Keywords: glioblastoma multiforme; C6 glioma; monoclonal antibodies; radioimmunolocalisation; targeted delivery

Vitamin B1: Metabolism and functions by A. F. Makarchikov (116-128).
The review highlights metabolism and biological functions of vitamin B 1 (thiamine). It considers thiamine transport systems in various organisms enzymes of its biosynthesis and degradation, as well as molecular basis of thiamine-dependent hereditary pathologies. A special attention is paid to discussion of the role of thiamine triphosphate and adenylated thiamine triphosphate, a new thiamine derivative recently discovered in living cells.
Keywords: vitamin B1; thiamine; thiamine phosphates; transport; metabolism; biological role

During the last 10–15 years significant progress has been achieved in all directions of studies of the glutathione system. A series of new enzymes involved into metabolism of glutathione has been discovered. Many of these enzymes are polyfunctional and their new activities have been recognized. The enzymes interact with hormones and signal transduction systems. Significant progress has been achieved in the studies of intracellular, intercellular and inter-organ transport. The important achievement is employment of not only selective compounds-analyzers but also gene engineering methods for identification of new functions.
Keywords: glutathione; metabolism enzymes; regulation

Insertion of exogenous ubiquitin into rat brain mitochondria in the presence of ATP and the ATPregenerating system (creatine phosphate/creatine phosphokinase) results in the increase in: sensitivity of mitochondrial monoamine oxidases (MAO) A and B to inhibition by mechanism based inhibitor and incorporation of [3H]-pargyline, which was especially notable in the fraction obtained by immunoprecipitation of mitochondrial proteins with anti-ubiquitin antiserum and protein A Sepharose. This suggests that MAO is a potential substrate for ubiquitination in vitro. However, the content of the tritium label in this fraction was less than 0.1 % and not more than 0.25% of total radioactivity of [3H]-pargyline bound to control and ATP-ubiquitin treated mitochondria, respectively. Insertion of ubiquitin into mitochondria did not influence molecular masses of [3H]-pargyline labeled proteins. These results suggest that direct ubiquitination of MAO insignificantly contributes to marked changes in the sensitivity of MAO A and MAO B to proteolysis and specific inhibition found under these experimental conditions. It is possible that more complex processes are involved into realization of these effects during ATP-dependent ubiquitin incorporation into mitochondria.
Keywords: monoamine oxidase; ubiquitin; proteolytic degradation; rat brain mitochondria; pargyline

Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila melanogaster by S. V. Sarantseva; O. I. Bolshakova; S. I. Timoshenko; A. A. Kolobov; M. P. Vitek; A. L. Schwarzman (149-155).
The phenomenon of protein transduction represents internalization of short peptides known as protein transduction domains (PTD) by cells. It is widely used in the development of new preparations for treatment of various brain disorders. However, the drug discovery process is limited by lack of simple and reliable models of blood brain barrier (BBB). These models should meet two main criteria: they should be applicable for testing of large numbers of samples simultaneously reproduce the physiological and functional characteristics of mammalian (including) human BBB. The major goal of this study was to estimate the BBB-crossing ability of known PTD-peptides using Drosophila melanogaster BBB as the model. We demonstrate here that after abdominal administration the PTD-peptide penetratin, derived from a Drosophila Antennapedia homeodomain protein can cross Drosophila and deliver the apoE mimetic peptide exhibiting neuroprotective properties.
Keywords: blood-brain barrier; transport; Drosophila melanogaster ; Antennapedia homeodomain; penetratin; protein transduction domain; apolipoprotein E mimetic peptides; biotin

The effect of thapsigargicin (TGC), a non-phorbol ester type tumor promoter, on Ca2+ movements has been investigated using L1210 mouse lymphoma cells. Ca2+ release from intact and digitonin permeabilized cells was evaluated using Fura-2 and Fura-3. TGC like Thapsigargin (TG) has the ability to discharge the intracellular Ca2+ stores and to increase intracellular free Ca2+ concentrations. TGC in a concentration dependent manner (0.16–16 nM) also inhibited cell growth and this effect was at least partially reversed by arachidonate.
Keywords: calcium; intracellular calcium; L1210 mouse lymphoma cells; thapsigargicin; thapsigargin

The extraction effect of ultrasound during plasma clot disruption by E. A. Chernyavsky; I. E. Adzerikho; V. M. Shkumatov (164-171).
The products of the plasma clot destruction by the low-frequency ultrasound (US) were analyzed using the combination of SDS gel-electrophoresis, gel filtration chromatography and scanning electron microscopy. It was found that US (27 kHz) did not cause activation of the plasmin system or covalent bonds cleavage in the fibrin molecules. At US intensities less than 21.6 W/cm2 there was extraction of blood serum proteins, which are located in the pores of the fibrin network. The increase in intensity of ultrasonic action resulted in protofibril dissociation, which was accompanied by further release into the solution of the blood serum proteins, located inside fibrin fibers. After US cavitation protein extracted from the plasma clot underwent aggregation. Interaction between free protofibrils resulted in formation of insoluble fibrin particles.
Keywords: ultrasound; plasma clot; fibrin; thrombolysis

Catalytic properties of glutathione reductase from liver at norm and toxic hepatitis by A. A. Agarkov; T. N. Popova; A. V. Semenikhina (172-176).
Catalytic properties of glutathione reductase (GR; EC 1.6.4.2) have been investigated using homogenous preparations of this enzyme purified from livers of control rats and rats with toxic hepatitis. Some properties of this enzyme remained unchanged under conditions of toxic hepatitis; these included eletrophoretic mobility (Rf = 0.23 ± 0.01), molecular mass (104.5 ± 5.2 kDa), pH optimum (7.4 ± 0.37), as well as close pK values of functional groups. However, enzyme isolated from toxic liver was characterized by lower affinity for substrate and coenzyme as well as by appearance of substrate inhibition. In addition there are some differences in regulation of GR activity by metabolites of tricarboxylic acid cycle.
Keywords: glutathione reductase; toxic hepatitis; properties; activity regulation

Antigenicity and B-epitope mapping of hepatitis C virus envelope protein E2 by T. I. Kuzmina; L. V. Olenina; M. A. Sanzhakov; T. E. Farafonova; T. V. Abramihina; J. Dubuisson; B. N. Sobolev; E. F. Kolesanova (177-182).
Immunogenicity for laboratory animals (rabbits and mice) of the whole hepatitis C virus envelope proteins and their conserved as well as hypervariable HVR1 sites has been investigated. Rabbit immune responses to HCV envelope proteins (both single E2 and E1E2 heterodimer) were shown to be much more efficient than murine immune responses. Rabbit immunization with E2 protein caused formation of antibodies to several highly conserved linear B-epitopes of this protein as well as to the N-terminal fragment of the hypervariable region HVR1. Epitopes in the CR2 region were determined for the first time. There was cross-reactivity between the N-terminal fragment of the protein E2 hypervariable region HVR1 and the octapeptide fragment of the protein E1 conserved region CR1, which shared four identical amino acid residues.
Keywords: hepatitis C virus; envelope proteins; immune response; antibodies; B-epitopes

Productive and non-productive complexes in cytochrome P450-containing systems by Yu. D. Ivanov; A. V. Ivanov; A. L. Kaysheva; V. G. Zgoda; S. A. Usanov; G. Hui-Bon-Hoa; A. I. Archakov (183-197).
The equilibrium dissociation constants KD, the complex association / dissociation rate constants (k on /k off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ lT ) to time required for a single hydroxylation cycle (τturnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τturnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.
Keywords: cytochrome P450 2B4; cytochrome P450scc; cytochrome P450cam; optical biosensor

Antitumor activity of L-asparaginase from Yersinia pseudotuberculosis by O. Yu. Abakumova; O. V. Podobed; A. A. Borisova; K. V. Sidoruk; S. S. Alexandrova; N. M. Omelyanuk; M. V. Pokrovskaya; L. I. Kondakova; N. N. Sokolov (198-201).
The cytotoxic activity of L-asparaginases from Yersinia pseudotuberculosis and from Erwinia carotovora were investigated in vitro using human T-lymphoblastic leukemia (Jurkat and Molt-4) and also solid tumor cell lines MCF-7 (human breast adenocarcinoma), LnCap (human prostate carcinoma), NGUK1 (rat Gasser node neurinoma). E.coli L-asparaginase produced by Medak (Germany) was used as a reference preparation. The data obtained indicate that Y. pseudotuberculosis L-asparaginase significantly inhibits growth of leukemic and solid tumor cells. Its antitumor activity is comparable to that of the reference preparation of L-asparaginase (Medak). These results suggest that the recombinant L-asparaginase can be used for the development of new preparations for the therapy of different types of tumors.
Keywords: L-asparaginase; tumor cells; cytotoxicity; Yersinia pseudotuberculosis

The decline in antibodies to hepatitis C virus during antiviral therapy by L. I. Nikolaeva; V. V. Makashova; E. V. Petrova; G. A. Shipulin; E. I. Samokhvalov; A. K. Tokmalaev; D. K. Lvov (202-209).
The goal of the study was to analyze B-cell response to hepatitis C virus during antiviral therapy among responders and non-responders. The content of antibodies to individual structural and non-structural HCV proteins was investigated during two years in three groups of patients: initial responders, non-responders and a reference group (without therapy). Treated patients in all groups exhibited the decrease in antibodies to analyzed HCV proteins, but with different patterns. The first statistically significant differences in the decline of the virus-specific antibodies between initial responders and non-responders were observed within the first three months after the beginning of therapy. Some treated patients demonstrated the decrease in antibody levels to HCV proteins after the end of therapy.
Keywords: hepatitis C; therapy; antibody