Biochemistry (Moscow) (v.76, #4)

Nonopioid effect of β-endorphin by Yu. A. Kovalitskaya; E. V. Navolotskaya (379-393).
This review presents the generalized literature data and the results of our own research of the nonopioid effect of β-endorphin, an opioid neuropeptide interacting not only with opioid but also with nonopioid (insensitive to the opioid antagonist naloxone) receptors. The roles of the hormone and its receptors in regulation of the immune, nervous, and endocrine systems are discussed. The effect of neuromediator on the immune system mediated by both opioid and nonopioid receptors is considered in detail. The data on distribution and function of the nonopioid β-endorphin receptor in human and animal organisms are presented. All available data on the characteristics of the nonopioid β-endorphin receptor obtained by means of radioligand analysis are given. The discussed information is supposed to extend our conceptions of the role of β-endorphin in mammals and to be of extensive use in medicine and pharmacology.
Keywords: β-endorphin peptides; naloxone-insensitive receptor; immune system; nervous system; adrenal glands

Cytokine receptor molecules have been shown to have extracellular domains of complex structure and a multistep activation system. Glycoprotein gp130 is a typical transducer of cytokine signal; it functions by forming multicomponent receptor complexes and transferring signals of tens of cytokines from the IL-6 family. Structural organization and basic functioning principles of gp130 are well known, as well as related signal pathways, which function during normal differentiation and are involved in pathogenesis of many tumors. The role of gp130 in IL-6-dependent tumors is best studied. In this review, based on extensive accumulated data, we examine the functional significance of certain parts of gp130 extracellular domains. Potentials of a recently developed method for estimation of receptor activation at the level of epitope structure are discussed.
Keywords: gp130 activation; cytokine; immunophenotyping; monoclonal antibodies; receptor structure; IL-6; cytokine-binding homology region

Mechanism of the Nrf2/Keap1/ARE signaling system by V. O. Tkachev; E. B. Menshchikova; N. K. Zenkov (407-422).
Nrf2 regulates expression of genes containing antioxidant-respons(iv)e element (ARE) in their promoters and plays a pivotal role among all redox-sensitive transcription factors. Nrf2 is constitutively controlled by repressor protein Keap1, which acts as a molecular sensor of disturbances in cellular homeostasis. These molecular patterns are in close inter-connection and function as parts of the integrated redox-sensitive signaling system Nrf2/Keap1/ARE. Depending on cellular redox balance, activity of this signaling system changes at the levels of transcription, translation, posttranslational modification, nuclear translocation of transcription factor, and its binding to ARE-driven gene promoters. This review summarizes current conceptions of Nrf2/Keap1/ARE induction and inactivation.
Keywords: redox regulation; Nrf2; Keap1; antioxidant-respons(iv)e element (ARE)

Effect of glutathione on growth of the probiotic bacterium Lactobacillus reuteri by KiBeom Lee; Ho-Jin Kim; Beom-Seop Rho; Sang-Kee Kang; Yun-Jaie Choi (423-426).
Glutathione (GSH) is an abundant nonprotein thiol that plays numerous roles within the cell. Previously, we showed that Lactobacillus salivarius has the capacity to mount a glutathione-mediated acid-tolerance response. In the present work we provide evidence of a requirement for GSH by Lactobacillus reuteri and have studied the role of GSH during cell growth. Medium supplementation with 0.5 mM GSH as the sole sulfur source enhanced cell growth, resulting in an increase in glucose consumption, and increased cell GSH and protein contents compared with levels seen in the absence of supplementation. Moreover, L. reuteri showed enhanced amino acid consumption when grown with 0.5 mM GSH. These findings indicate that glutathione is a nutrient for bacterial growth.
Keywords: amino acid consumption; bacterial growth; glutathione; Lactobacillus reuteri ; protein content

The rate of PSI mediated cyclic electron transport was studied in wild type and mutant cells of Synechocystis sp. PCC 6803 deficient in NDH-1 (M55) or succinate dehydrogenase (SDH) that are responsible for the dark reduction of the plastoquinone pool. Kinetics of P700 photooxidation and P700+ dark reduction in the presence of 5·10−5 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea have been registered as light induced absorbance changes at 810 nm resulting from illumination of cells with 730-nm actinic light for 1 sec. It is shown that in the absence of dehydrogenases the rate of dark reduction of P700+ in both mutants did not decrease but even increased in NDH-1-less mutant cells as compared with the rate in wild type cells. Dibromothymoquinone drastically reduced the rate of P700+ dark reduction both in wild type and in mutant cells. Thus, the cyclic electron transfer from ferredoxin through the plastoquinone pool to P700+, which is independent from dehydrogenases, takes place in all the types of cells. Preillumination of cells of wild type and both mutants for 30 min or anaerobic conditions resulted in delay of P700 photooxidation and acceleration of P700+ dark reduction, while the level of photosynthesis and respiration terminal acceptors (NAD(P)+ and oxygen) decreased. It appears that the rate of P700 photooxidation and P700+ dark reduction in cyclic electron transport in Synechocystis wild type and mutant cells is determined by the level of NADP+ and oxygen in stroma. A possible approach to evaluation of the levels of these acceptors in vivo is proposed, based on kinetic curve parameters of P700 photoconversions induced by 730-nm light with 1-sec duration.
Keywords: cyclic electron transport; NADPH; P700 redox transients; NDH-1; SDH

A lectin was purified from fruit bodies of the milk mushroom Lactarius pergamenus (Fr.) Fr. by a combination of ethanol precipitation, affinity chromatography on copolymer of polyvinyl alcohol and human blood B-group-specific sub-stance, and ion-exchange chromatography on DEAE-Toyopearl. The lectin yield was 3 mg/kg of fresh mushrooms. Considerable loss of primary activity was observed during its purification, which, presumably, could be explained by disin-tegration of the lectin molecule, which consisted of six subunits, first to two molecules of three subunits, and then to individual subunits. There was a reverse tendency to aggregation during concentration of lectin solutions. Similar processes can take place in nature because of considerable individual variations of the lectin activity during growth of mushroom fruit bodies. The lectin weakly interacts with DGalNAc, while DGalβ1-3DGalNAc and DGalβ1-3DGlcNAc are the most probable candidates for ligands, with which the L. pergamenus lectin interacts at disaccharides level. The purified lectin may find application in histochemical research.

Histidine M182 in the reaction center (RC) of Rhodobacter sphaeroides serves as the fifth ligand of the bacterio-chlorophyll (BChl) BB Mg atom. When this His is substituted by an amino acid that is not able to coordinate Mg, bacterio-pheophytin appears in the BB binding site instead of BChl (Katilius, E., et al. (1999) J. Phys. Chem. B, 103, 7386–7389). We have shown that in the presence of the additional mutation I(L177)H the coordination of the BChl BB Mg atom in the double mutant I(L177)H+H(M182)L RC still remains. Changes in the double mutant RC absorption spectrum attributed to BChl absorption suggest that BChl BB Mg atom axial ligation might be realized not from the usual α-side of the BChl macrocycle, but from the opposite, β-side. Weaker coordination of BChl BB Mg atom compared to the other mutant RC BChl molecules suggests that not an amino acid residue but a water molecule might be a possible ligand. The results are discussed in the light of the structural changes that occurred in the RC upon Ile/His substitution in the L177 position.
Keywords: bacterial photosynthesis; Rhodobacter sphaeroides ; photosynthetic reaction center; site-directed mutagenesis; bacteriochlorophyll; Mg coordination

Heat shock protein DnaK — Substrate of actin-specific bacterial protease ECP32 by A. V. Morozova; S. Yu. Khaitlina; A. Yu. Malinin (455-461).
It has been found that actin-specific bacterial protease ECP32 cleaves prokaryotic heat shock protein DnaK, which belongs to the family of heat shock proteins with molecular weight 70 kDa. We propose a new one-step method for DnaK purification using heat treatment. The technique yields ∼1 mg of partially purified DnaK from 25 g of wet bacterial biomass. Polyclonal antibodies against DnaK were obtained. The degree of ECP32 catalyzed proteolysis of partially purified DnaK and that of DnaK in initial cell extracts was compared.
Keywords: protease ECP32; DnaK; protein purification; limited proteolysis

Correlation between influence of polysaccharides on hydrolase activity and their antiviral effect in tobacco leaves by L. A. Lapshina; V. P. Nagorskaya; A. V. Reunov; A. O. Barabanova; N. M. Shevchenko; I. M. Yermak; T. N. Zvyagintseva; L. A. Elyakova (462-466).
The activities of hydrolases (acid phosphatase, RNase, and proteases) in healthy and tobacco mosaic virus-infected leaves of Nicotiana tabacum L. var. Samsun, both untreated and treated with polysaccharides (PS) (1,3;1,6-β-D-glucan, fucoidan, and κ/β-carrageenan), were determined. The PS lead to substantial increase in the hydrolase level. The percentage of viral particles undergoing destructive change also increases in leaves treated with PS 24 h before infection. We suppose that the PS-mediated hydrolase activation promotes intracellular destruction of the viral particles and, thus, comprises one of the PS-induced protective mechanisms limiting intracellular viral accumulation.
Keywords: 1,3;1,6-β-D-glucan; fucoidan; κ/β-carrageenan; acid phosphatase; RNase; protease; tobacco mosaic virus

Effect of homocysteine on properties of neutrophils activated in vivo by E. A. Bryushkova; E. A. Vladychenskaya; M. S. Stepanova; A. A. Boldyrev (467-472).
We have found that neutrophils begin to express NMDA receptors on their membranes after in vivo activation. These receptors are the target for action of homocysteine (HC). After incubation of activated neutrophils with HC, the degranulation process is stimulated and generation of reactive oxygen species is increased. We conclude that expression of NMDA receptors on neutrophil membrane makes neutrophils sensitive to HC. Thus, hyperhomocysteinemia may induce additional stimulation of immune competent cells.
Keywords: neutrophils; inflammation; NMDA receptors; homocysteine

Cyanines as efficient photosensitizers in photodynamic reaction: Photophysical properties and in vitro photodynamic activity by J. Kulbacka; A. Pola; D. Mosiadz; A. Choromanska; P. Nowak; M. Kotulska; M. Majkowski; A. Hryniewicz-Jankowska; L. Purzyc; J. Saczko (473-479).
The purpose of the present study was to explore the potential application of cyanines in photodynamic treatment. The photophysical features of four cyanines (KF570, HM118, FBF-749, and ER-139) were investigated by elemental and spectral analyses. Two malignant cell lines (MCF-7/WT and MCF-7/DOX) were used to test the potential for use in the photodynamic therapy. The cytotoxic effects of these dyes were determined by the MTT assay after 4 and 24 h of incubation with the cyanine. KF570 and HM118 were irradiated with red light (630-nm filter) and FBF-749 and ER-139 with green light (435-nm filter). The results showed that the cyanine HM118 demonstrated a major phototoxic effect. It was also noted that the efficiency of photodynamic therapy was higher in the doxorubicin-resistant cell line (MCF-7/DOX).
Keywords: breast adenocarcinoma cells; cyanines; photodynamic therapy; cytotoxicity

Oligopeptidase B from Serratia proteamaculans. II. Enzymatic characteristics: Substrate analysis, influence of calcium ions, pH and temperature dependences by A. G. Mikhailova; R. F. Khairullin; I. V. Demidyuk; T. Yu. Gromova; S. V. Kostrov; L. D. Rumsh (480-490).
Enzymatic properties of a novel oligopeptidase B from psychrotolerant gram-negative microorganism Serratia proteamaculans (PSP) were studied. The substrate specificity of PSP was analyzed using p-nitroanilide substrates, and the influence of calcium ions on the enzyme activity was studied. Hydrolysis of oligopeptides by PSP was studied using melittin as the substrate. Optimal conditions for the PSP activity (pH and temperature) have been established. It was found that PSP shares some properties with oligopeptidases B from other sources containing two Asp/Glu residues in the S2 site, but it differs significantly in some characteristics. The S2 site of PSP contains only one Asp460 residue. The secondary specificity of PSP has a number of specific features: an unusual substrate inhibition by peptides with hydrophobic residues at the P2 position, as well as the drastic influence of calcium ions on substrate characteristics of the enzyme. It is assumed that the PSP molecule contains a large hydrophobic substrate-binding site, and significant conformational rearrangements of the enzyme active site are induced by Ca2+ binding and by the formation of the enzyme-substrate complex. The temperature characteristics of PSP (high activity at low temperature as well as low apparent temperature optimum (25°C)) confirm that PSP is a psychrophilic enzyme.
Keywords: oligopeptidase B; Serratia proteamaculans ; psychrophilic enzymes; substrate analysis; melittin; pH dependence

The possible application of the quasi-equilibrium assumption for an arbitrary mechanism of enzymatic reaction is considered. It is shown at what ratios of kinetic constants a segment consisting of two, three, and four intermediates may be considered as an equilibrium one. Expressions for evaluation of accuracy of distribution of intermediate concentrations inside the equilibrium segment and accuracy of determination of intermediate concentrations inside and outside the equilibrium segment as a function of the ratio of kinetic constants are derived. A method for determination of the limitations on the ratio of rate constants for an equilibrium segment of arbitrary structure is suggested.
Keywords: enzyme kinetics; quasi-equilibrium assumption; accuracy of assumption; Cha method; rapid equilibrium; steady-state kinetics; graph method

Cellular and Molecular Biology of Metals by G. Ya. Wiederschain (493-493).

Data mining techniques for the life sciences by G. Ya. Wiederschain (494-494).