Biochemistry (Moscow) (v.73, #4)

Mechanisms determining phenotypic heterogeneity of hepatocytes by I. F. Usynin; L. E. Panin (367-380).
This review summarizes results of biochemical and immunohistochemical studies indicating the existence of functional heterogeneity of hepatocytes depending on their localization in the hepatic acinus; this determines characteristic features of metabolism of carbohydrates, lipids, and xenobiotics. The physiological significance of hepatocyte heterogeneity is discussed. According to the proposed model of intercellular communication, the metabolic specialization of hepatocytes is determined by secretory activity of hepatic resident macrophages (Kupffer cells) localized mainly in the periportal zone of the liver acinus. Macrophages participate in secretion of a wide spectrum of intercellular mediators (cytokines, prostaglandins, growth factors) and also in metabolism of numerous blood metabolites and biologically active substances (hormones, lipoproteins, etc.). In the sinusoid and in the space of Disse (also known as perisinusoidal space) they form a concentration gradient of regulatory factors and metabolites inducing the phenotypic differences between hepatocytes.
Keywords: liver; metabolism; hepatocyte; heterogeneity; macrophage; Kupffer cell; intercellular communications

Tear proteins in health, disease, and contact lens wear by R. Sariri; H. Ghafoori (381-392).
Although well known as manifestations of sorrow, emotions, frustration, and blackmail, tears have a more prosaic and important function as a lubricant and as a blood substitute for the cornea. Tears transport oxygen and carbon dioxide and play a central role in the cellular economy of the ocular surface and conjunctiva. In addition to proteins, tears contain lipids and glycoproteins, which increase the wetting effect of the aqueous component and delay evaporation. The total protein concentration of tears is about 10% of that of the plasma. About 80 proteins and polypeptide components have been detected by electrophoresis. Among 30 proteins identified in tears, about 50% are enzymes. Some of the tear enzymes are secreted by the lacrimal glands; others are produced by or released from epithelial cells of the cornea and the conjunctiva. Finally, a few enzymes originate from plasma and appear in tears only in cases with increased permeability of the conjunctival vessels. The aim of this review is to provide clinical and biochemical information about tear enzymes both for ophthalmologists and for biochemists interested in clinical and experimental tear enzymology.
Keywords: tear enzymes; lysozyme; lactoferrin; tear specific pre-albumin; contact lenses; eye disorders

Mammalian galectins: Structure, carbohydrate specificity, and functions by E. M. Rapoport; O. V. Kurmyshkina; N. V. Bovin (393-405).
Galectins are a family of β-galactoside binding lectins, homological by a sequence of the carbohydrate-binding site. In this review literature data about structure and carbohydrate specificity of galectins are discussed. The role of galectins in the regulation of cell adhesion in immune response, inflammation, and cancer progression is considered.
Keywords: apoptosis; galectins; cell adhesion; lactosamine; carbohydrate specificity

Structure and characterization of the gene cluster of the O-antigen of Escherichia coli O49 containing 4,6-dideoxy-4-[(S-3-hydroxybutanoylamino]-D-glucose by A. V. Perepelov; Quan Wang; S. N. Senchenkova; S. D. Shevelev; A. S. Shashkov; Lu Feng; Y. A. Knirel; Lei Wang (406-410).
An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of enteropathogenic Escherichia coli O49 and studied by sugar analysis along with one- and two-dimensional 1H- and 13C-NMR spectroscopy. The following structure of the linear tetrasaccharide repeating unit of the O-polysaccharide was established: $$ o 2) - alpha - D - Quip4N(S3HOBut) - (1 o 4) - eta - D - GalpNAc - (1 o 4) - alpha - L - Rhap - (1 o 3) - eta - D - GlcpNAc6Ac - (1 o , $$ where d-Qui4N(S3HOBut) stands for 4,6-dideoxy-4-[(S)-3-hydroxybutanoylamino]-d-glucose and O-acetylation of GlcNAc is partial (∼30%). To our knowledge, no N-(3-hydroxybutanoyl) derivative of Qui4N has been hitherto found in bacterial polysaccharides. Gene functions of the O-antigen gene cluster of E. coli O49 were assigned by bioinformatics analysis and found to correspond to the O-polysaccharide structure. Two new genes were revealed and suggested to be responsible for synthesis and transfer of the 3-hydroxybutanoyl group.
Keywords: Escherichia coli ; lipopolysaccharide; bacterial polysaccharide structure; 3-hydroxybutyrate; 4-amino-4,6-dideoxy-d-glucose; O-antigen gene cluster

It is well known that at the beginning of mitosis the nucleolus disassembles but then reassembles at the end of mitosis. However, the mechanisms of these processes are still unclear. In the present work, we show for the first time that selective inhibition of cyclin B-dependent kinase 1 (CDK1) by roscovitine induces premature assembly of the nucleolus in mammalian cells in metaphase. Treatment of metaphase cells with roscovitine induces formation of structures in their cytoplasm that contain major proteins of the mature nucleolus participating in rRNA processing, such as B23/nucleophosmin, C23/nucleolin, fibrillarin, Nop52, as well as partially processed (immature) 46–45S pre-rRNA. This effect is reproducible in cells of various types; this indicates that general mechanisms regulate early stages of the nucleolus reassembly with CDK1 participation in mammalian cells. Based on our and literature data, we suggest that inactivation of the CDK1-cyclin B complex at the end of mitosis results in dephosphorylation of B23/nucleophosmin and C23/nucleolin; this facilitates their interaction with pre-rRNA and leads to formation of insoluble supramolecular complexes-nucleolus-derived foci.
Keywords: nucleolus; mitosis; cyclin B-dependent kinase CDK1; nucleolus-derived foci (NDF); fibrillarin; B23/nucleophosmin; roscovitine

The inhibitor of catalase 3-amino-1,2,4-triazole (AMT) was used to study the physiological role of catalase in the yeast Saccharomyces cerevisiae under starvation. It was shown that AMT at the concentration of 10 mM did not affect the growth of the yeast. In vivo and in vitro the degree of catalase inhibition by AMT was concentration- and time-dependent. Peroxisomal catalase in bakers’ yeast was more sensitive to AMT than the cytosolic one. In vivo inhibition of catalase by AMT in S. cerevisiae caused a simultaneous decrease in glucose-6-phosphate dehydrogenase activity and an increase in glutathione reductase activity. At the same time, the level of protein carbonyls, a marker of oxidative modification, was not affected. Possible mechanisms compensating the negative effects caused by AMT inhibition of catalase are discussed.
Keywords: Saccharomyces cerevisiae ; 3-amino-1,2,4-triazole; catalase; glucose-6-phosphate dehydrogenase

To achieve high throughput and economical format of 2-D PAGE, comparison between gel size and resolution was conducted on human breast carcinoma cell line (MCF-7/AZ) proteins. SDS gel length showed a weaker influence of separation length on resolution in the second dimension, and there was little benefit of separation distances greater than 15 to 19 cm. IPG strip separation distances were very important with dramatic increase in resolution of longer gels compared with smaller gels, and maximal resolution was obtained using 18-and 24-cm IPG strips. Loading optimal amount of proteins on 2-D gels can also increase the number of detected spots. Therefore, taken together, compromise 2-D gels are crucial for higher capacity and higher throughput.
Keywords: two-dimensional polyacrylamide gel electrophoresis; immobilized pH gradient (IPG) strip length; SDS gel length/high throughput; resolution

Interaction of chitosans and their N-acylated derivatives with lipopolysaccharide of gram-negative bacteria by G. A. Naberezhnykh; V. I. Gorbach; G. N. Likhatskaya; V. N. Davidova; T. F. Solov’eva (432-441).
The interactions of lipopolysaccharide (LPS) with the natural polycation chitosan and its derivatives—high molecular weight chitosans (80 kD) with different degree of acetylation, low molecular weight chitosan (15 kD), acylated oligochitosan (5.5 kD) and chitooligosaccharides (biose, triose, and tetraose)—were studied using ligand-enzyme solid-phase assay. The LPS-binding activity of chitosans (80 kD) decreased with increase in acetylation degree. Affinity of LPS interaction with chitosans increased after introduction of a fatty acid residue at the reducing end of chitosan. Activity of N-monoacylated chitooligosaccharides decreased in the order: oligochitosan → tetra-→ tri-→ disaccharides. The three-dimensional structures of complexes of R-LPS and chitosans with different degree of acetylation, chitooligosaccharides, and their N-monoacylated derivatives were generated by molecular modeling. The number of bonds stabilizing the complexes and the energy of LPS binding with chitosans decreased with increase in acetate group content in chitosans and resulted in changing of binding sites. It was shown that binding sites of chitooligosaccharides on R-LPS overlapped and chitooligosaccharide binding energies increased with increase in number of monosaccharide residues in chitosan molecules. The input of the hydrophobic fragment in complex formation energy is most prominent for complexes in water phase and is due to the hydrophobic interaction of chitooligosaccharide acyl fragment with fatty acid residues of LPS.
Keywords: lipopolysaccharide; chitosan; binding; computer simulation; molecular docking

Novel DNA glycosylases from Mycobacterium tuberculosis by V. S. Sidorenko; M. A. Rot; M. L. Filipenko; G. A. Nevinsky; D. O. Zharkov (442-450).
Oxidized bases are removed from DNA of Escherichia coli by enzymes formamidopyrimidine DNA glycosylase (Eco-Fpg) and endonuclease VIII (Eco-Nei) of the same structural family Fpg/Nei. New homologs of these enzymes not characterized earlier have been found in genomes of Actinobacteria. We have cloned and expressed two paralogs (Mtu-Nei2 and Mtu-Fpg2) from 36KAZ and KHA94 isolates of Mycobacterium tuberculosis and studied their ability to participate in DNA repair. Under heterologous expression in E. coli, Mtu-Nei2 decreased the rate of spontaneous mutagenesis in the rpoB gene, whereas Mtu-Fpg2 moderately increased it, possibly due to absence of residues crucially important for catalysis in this protein. Mtu-Nei2 was highly active toward double-stranded DNA substrates containing dihydrouracil residues and apurine-apyrimidine sites and was less efficient in cleavage of substrates containing 8-oxoguanine and uracil residues. These lesions, as well as 8-oxoadenine residues, were also recognized and removed by the enzyme from single-stranded DNA. Fpg and Nei homologs from M. tuberculosis can play an important role in protection of bacteria against genotoxic stress caused by oxidative burst in macrophages.
Keywords: oxidative stress; DNA repair; DNA glycosylases; Mycobacterium tuberculosis

Immune proteasomes in the developing rat thymus by V. I. Melnikova; M. A. Afanasieva; S. B. Dmitrieva; Ya. D. Karpova; N. P. Sharova; L. A. Zakharova (451-457).
The age dynamics of the content of the immune proteasome subunits LMP2 and LMP7 in rat thymus during prenatal and early postnatal ontogeny was studied. The LMP2 and LMP7 immune subunits were detected by Western blotting already by the 18th day of embryonic development, their amount increased to the 21st day to the level characteristic of the postnatal state. Double immunofluorescent labeling showed that in the thymus tissue the largest amount of LMP2 and LMP7 is localized in epithelial cells, whereas the level of their expression in thymocytes is lower. The results suggest that the establishment in thymus of selection processes, which depend on activity of immune proteasomes, can take place already in prenatal ontogeny. Analysis of age dynamics of the natural apoptosis level in thymocytes also favors this supposition. The presence of immune proteasomes in thymocytes during perinatal ontogeny suggests that, besides the antigen presentation, immunoproteasomes may possess other important functions.
Keywords: immune subunits of proteasomes; thymus; ontogeny; negative selection; thymic epithelial cells; apoptosis; rat

Using steady-state, polarized, and phase-modulation fluorometry, the dithiothreitol-induced denaturation of insulin and formation of its complex with α-crystallin in solution were studied. Prevention of the aggregation of insulin by α-crystallin is due to formation of chaperone complexes, i.e. interaction of chains of the denatured insulin with α-crystallin. The conformational changes in α-crystallin that occur during complex formation are rather small. It is unlikely that N-termini are directly involved in the complex formation. The 8-anilino-1-naphthalenesulfonate (ANS) is not sensitive to the complex formation. ANS emits mainly from α-crystallin monomers, dimers, and tetramers, but not from oligomers or aggregates. The possibility of highly sensitive detection of aggregates by light scattering using a spectrofluorometer with crossed monochromators is demonstrated.
Keywords: α-crystallin; insulin; chaperone complex; aggregation; light scattering; fluorescence

Participation of oxygen in the bacterial transformation of 2,4,6-trinitrotoluene by E. A. Naumenko; A. V. Naumov; E. S. Suvorova; R. Gerlach; A. M. Ziganshin; A. P. Lozhkin; N. I. Silkin; R. P. Naumova (463-469).
The exposure of Bacillus cereus ZS18 cell suspensions to 2,4,6-trinitrotoluene (TNT) in the absence of other oxidizable substrates increases oxygen uptake, exceeding the basal level of respiration of the bacterium 1.5- and 2-fold with 50 and 100 mg/liter of TNT, respectively. The interaction of both living and to less extent dead bacterial cells with TNT results in the accumulation of superoxide anion ( $$ O_2^{dot ---} $$ ) in the extracellular medium, which was revealed by the EPR spectroscopy. The accumulation of $$ O_2^{dot ---} $$ decreased by 50–70% in the presence of Cu,Zn-superoxide dismutase of animal origin. In the presence of living bacterial cells, the level of TNT decreased progressively, yielding hydroxylaminodinitrotoluenes together with $$ O_2^{dot ---} $$ . In the presence of heat-killed cells, a moderate decrease in TNT was observed, and the appearance of $$ O_2^{dot ---} $$ was not accompanied by the production of any detectable TNT metabolites. Chelating agents inhibited the transformation of TNT and decreased the formation of $$ O_2^{dot ---} $$ . The demonstrated generation of $$ O_2^{dot ---} $$ during the interaction of TNT with K4[Fe(CN)6] together with the observed effects of chelating agents suggest the participation of iron in the one-electron reduction of TNT and the functioning of an extracellular redox cycle with the involvement of molecular oxygen.
Keywords: 2,4,6-trinitrotoluene; aerobic transformation; superoxide anion

Generation of hydrogen peroxide and hydroxyl radicals in L-amino acid solutions in phosphate buffer, pH 7.4, under X-ray irradiation was determined by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system and using the fluorescent probe coumarin-3-carboxylic acid, respectively. Amino acids are divided into three groups according to their effect on the hydrogen peroxide formation under irradiation: those decreasing yield of H2O2, having no effect, and increasing its yield. All studied amino acids at 1 mM concentration decrease the yield of hydroxyl radicals in solution under X-ray irradiation. However, the highest effect is observed in the order: Cys > His > Phe = Met = Trp > Tyr. At Cys, Tyr, and His concentrations close to physiological, the yield of hydroxyl radicals decreases significantly. Immunoenzyme analysis using monoclonal antibodies to 8-oxoguanine (8-oxo-7,8-dihydroguanine) was applied to study the effect of amino acids with the most pronounced antioxidant properties (Cys, Met, Tyr, Trp, Phe, His, Lys, Arg, Pro) on 8-oxoguanine formation in vitro under X-ray irradiation. It is shown that amino acids decrease the content of 8-oxoguanine in DNA. These amino acids within DNA-binding proteins may protect intracellular DNA against oxidative damage caused by formation of reactive oxygen species in conditions of moderate oxidative stress.
Keywords: amino acids; X-ray irradiation; hydrogen peroxide; hydroxyl radicals; antioxidants; 8-oxoguanine in DNA

Experimental testing of the role of cytoskeleton in the solution by neurons of problems facing the brain by E. A. Liberman; S. V. Minina; D. A. Moshkov; I. M. Santalova; I. A. Chistopolskiy; N. E. Shklovski-Kordi (479-482).
Investigation of the influence of cAMP on neuronal electric activity suggests that nerve cells can solve problems using an intraneuronal calculating medium based on the cytoskeleton. When a new problem is posed, this structure has to be disassembled and assembled by the neuronal molecular computer according to the program recorded in DNA. If DNA lacks an appropriate program, the cytoskeleton will not be assembled. In our experiments, fishes which were rotated simultaneously around two mutually perpendicular axes lost their swimming ability, and some dramatic changes were observed in the cytoskeleton of their Mauthner neurons. These changes disappeared after a long-term rest: the cytoskeleton was restored simultaneously with the ability for normal swimming.
Keywords: molecular quantum generator; cytoskeleton; calculating medium

Fundamentals of Biochemical Calculations, 2nd Edn. by G. Ya. Wiederschain (483-483).