Construction of a Fusion Enzyme Exhibiting Superoxide Dismutase and Peroxidase Activity.

A chimeric gene construct encoding human peroxiredoxin 6 and Mn-superoxide dismutase from Escherichia coli was developed. Conditions for expression of the fusion protein in E. coli cell were optimized. Fusing of the enzymes into a single polypeptide chain with peroxiredoxin 6 at the N-terminus (PSH) did not affect their activities. On the contrary, the chimeric protein with reverse order of enzymes (SPH) was not obtained in a water-soluble active form. The active chimeric protein (PSH) exhibiting both peroxidase and superoxide dismutase activities was prepared and its physicochemical properties were characterized.

[Xenopus laevis peroxiredoxins: Gene expression during development and characterization of the enzymes].

Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1-6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10-63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1-6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1-6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.

Cardioprotective Effect of Modified Peroxiredoxins in Retrograde Perfusion of Isolated Rat Heart under Conditions of Oxidative Stress.

Antioxidant properties of recombinant peroxiredoxin-6 and chimeric protein PSH combining peroxidase and superoxide dismutase activities were studied on the model of retrograde perfusion of isolated rat heart under conditions of H2O2-induced oxidative stress. The exogenous antioxidant proteins exhibited cardioprotective properties manifested in heart rate normalization, maintenance of contractile activity of the myocardium, and prevention of H2O2-induced LPO in oxidative stress. Localization of peroxiredoxin-6 and PSH in the cardiac tissue was determined and myocardial structures most effectively protected by the antioxidant enzymes from ischemia/reperfusion-induced damages were identified. The results suggest that modified peroxiredoxins are promising components of perfusion media for preservation of isolated organs.

[Peroxyredoxins as multifunctional enzymes].

Peroxiredoxins are evolutionarily ancient, but relatively recently discovered group of seleniumindependent peroxidases. Peroxiredoxins protect cells from various peroxides and play an important role in maintaining the oxidation-reduction homeostasis. Moreover, they are involved in many cellular processes that are not related to peroxidase activity. Here, recent data on the structure and function of peroxiredoxins, regulation of gene expression and activity of different peroxiredoxins are considered.

[Modified peroxiredoxins as prototypes of drugs a powerful antioxidant].

The possibility of using modified peroxyredoxins as powerful antioxidant agents has been considered. Peroxyredoxins immobilized on perfluorocarbon emulsions and PTD-modified peroxyredoxins have been studied. It has been shown that peroxyredoxins efficiently bind to particles of perfluorocarbon emulsions, while maintaining their antioxidant properties. A panel of PTD-modified peroxyredoxins has been created and peroxyredoxins most effective both in antioxidant properties and the ability to penetrate cells have been selected. The modified peroxyredoxins obtained may serve as the basis for the design of drug with powerful antioxidant action.

[Two peroxiredoxins 6 of Xenopus laevis].

Two different genes of peroxiredoxin 6 are encoded in the genome of Xenopus laevis: xen1 (Acc.no. EMBL Data Bank - BCO54278) and xen2 (Acc.no. EMBL Data Bank - BC540309). Both genes were cloned and expressed in Escherichi coil. Proteins were purified and analyzed. The amino acid sequences of the enzymes Xen1 and Xen2 are 95% identical with the same peroxidase activity, pH and temperature optimums, as well as thermostability, being approximately equal. The expression of peroxiredoxin 6 genes significantly differ during ontogenesis of X. laevis. The expression of xen1 starts on a later stage of development 47-48, while the gene xen2 is expressed on all stages of development with the same increase starting from stage 0-5. The level of xen2 expression in embryos increased after incubation in presence of hydrogen peroxide. The comparison of amino acid sequences of proteins Xen1 and Xen2 shows that only the enzyme Xen2 may have phospholipase activity, since it has residues of phospholipase A2 active center: Ser31, His25, Asp139.

Peroxiredoxin 6 from the clawed frog Xenopus laevis: cDNA cloning, enzyme characterization, and gene expression during development.

The Xenopus laevis 1-Cys-peroxiredoxin (peroxiredoxin 6, Prx6) gene was cloned and expressed in Escherichia coli. The enzymatic properties of the recombinant protein were characterized and compared to those of human Prx6. Xenopus laevis Prx6 has 224 amino acid residues including five Cys, one of which, Cys47, is located in the active center determining peroxidase activity. The stability and activity of X. laevis Prx6 relative to hydrogen peroxide and tret-butyl hydroperoxide are very similar to corresponding values for human Prx6. Both enzymes have temperature optimum at 37 degrees C, but the clawed frog enzyme retains no less than 50% of activity over a wider temperature interval (10-50 degrees C) than the human one (25-50 degrees C). The expression of X. laevis prx6 at different stages of development was investigated. The level of gene expression increased during development, especially at stages 33-43 during formation of the lungs, when heartbeat and hatching begins.

[Cloning, expression and comparative analysis of peroxiredoxine 6 from different species].

Human, rat, Xenopus and Drosophila (Dpx2540 and Dpx6005) cDNA of peroxiredoxins were cloned and expressed in Escherichia coli. Their enzymatic activity, temperature optimum and thermostability were determined. For H2O2 the activity of enzymes decreased in the following order: DPx2540 > > human > Xenopus >rat > DPx6005. For tret-butyl hydroperoxide the order of activity decrease is: DPx2540 = DPx6005 > rat > Xenopus > human. Effectiveness of plasmid DNA protection from oxidative damage mediated by Fenton reaction is: Dpx2540 > Dpx6005 = rat = human > Xenopus. The optimal temperature for activity of all these enzymes is 37 degrees C. Peroxiredoxins from rat, Xenopus and Drosophila (Dpx 6005) retain no less than 50% of activity in a wide temperature range (10-50 degrees C) in contrast to human and Drosophila (Dpx 2540) enzymes with the interval of only 25-45 degrees C. The thermostability of enzymes decreased in the following order: Dpx6005 > or = rat > human > Xenopus > Dpx2540. So, there is negative correlation between activity and stability of peroxiredoxin 6.

[Structural and functional analysis of the representatives of a new class of retroelements in Drosophila species].

Various copies of mobile element Penelope previously described in Drosophila virilis have been investigated in different strains of D. virilis and transgenic strains of D. melanogaster transformed by P-based constructs containing full-size copy of Penelope. It has been demonstrated that most of Penelope copies in both species carry large terminal inverted repeats (TIR) and contain deletions of various size at their 5'end of ORF. Junctions between TIR and ORF usually carry microhomology of various length enabling to postulate a hypothesis explaining the molecular mechanism underlying the origin of such complex structures. Penelope copies usually carry 34 bp repeat located in direct orientation at both end of ORF and target site duplications of various length.

Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures.

A new cloning vector pN15L is described. It is a linear 13.8 kb plasmid based on the coliphage N15 mini-replicon. The vector capacity exceeds 50 kb and the copy number is 250 per Escherichia coli chromosome. We show that some artificial and natural palindromes and approximately 5% of human DNA Bgl II fragments can be cloned effectively in linear vector pN15L, whereas they either sharply reduce the copy number of circular vector pUC19 or cannot be cloned at all. We conclude that pN15L may be usefully employed to clone large imperfect palindromes and some abnormal sequences of human DNA.

[COS-region of temperate coliphage N15]. / COS-raion umerennogo bakteriofaga N15.

The cohesive termini including the cos region (altogether 414 bp) of the DNA of the temperate coliphage N15 are sequenced. The termini are complementary 12-nucleotide single-stranded 5'-extended DNAs. The sequence of the left terminus is 5'-GGGCGGCGTCCG-3', that of the right 5'CGGACGCCGCCC-3'. Ten nucleotides of the N15 termini are identical to those of phage lambda. The N15 and lambda sequences are notably homologous only within the 50 bp region from the left and right ends. Phage N15 has a region with the nucleotide sequence identical to the R4 site of phage lambda, presumably reacting with terminase. This region is situated in the same site with regard to the cohesive sequence as in phage lambda. The cos region of N15 has no sequences similar to R1, R2, and R3 of lambda. N15 has a sequence similar to IHF of phage lambda, but in N15 this sequence is located near the right but not left (as in phage lambda) terminus. Computer analysis revealed palindromes and repeats within 450 bp of N15, including the cohesive termini.

[Cloning of large imperfect palindromes in circular and linear vectors]. / Klonirovanie krypnykh neideal'nykh palindromov v kol'tsevom i lineinom vektorakh.

Data on comparative analysis of cloning of large imperfect artificial palindromes and one natural palindrome in the circular pUC19 and linear pN15L vectors, constructed on the basis of temperate N15 bacteriophage minireplicon are presented. The artificial palindromes consisted of the two head-to-head oriented 5:5-kb lambda bacteriophage DNA fragments interrupted by a short sequence of varied size. Natural palindrome was represented by the 12.5-kb BamHI fragment of Tetrahymena pyriformis ribosomal genes cluster. Integration of some artificial palindromes and a natural palindrome into a circular vector resulted in a considerable decrease of its copy number. This was assumed to result from cruciform formation mediated by supercoiling of a circular vector DNA. Thus, a linear is vector preferable in cloning of inverted repeated DNA sequences.

Induction and repression of genes of the rat small intestine mucosa with excess and insufficiency of alimentary iron.

Three-month-old Wistar rats were fed with either an iron-deficient diet for 21 days or an iron-excessive diet for 10 days. Fifty thousand clones of a cDNA library of small intestine mucosa were hybridized with two radioactive samples synthesized on mRNA from the small intestine of rats fed with iron-excessive or iron-deficient diets. As a result, genes were found with mRNA level depending on the content of alimentary iron. Iron deficiency increased the mRNA level of genes of apolipoprotein AIV and class 1 antigen and decreased the mRNA level of the apolipoprotein AI gene and of an unknown gene, while no change was found in the mRNA level of the gene of fatty acid-binding protein. Excess of dietary iron resulted in the increase in mRNA of this gene and in a decrease in the apolipoprotein AI gene and in the unknown gene. The mRNA of apolipoprotein AIV did not change. The data indicate that changes in the level of alimentary iron influence the expression of genes involved in metabolism of lipids.

Modifying effect in vivo of interferon alpha on induction and repair of lesions of DNA of lymphoid cells of gamma-irradiated mice.

The induction of structural lesions and repair in DNA of lymphoid cells from the peripheral blood, spleen and thymus of mice treated with natural mouse interferon alpha (IFN-alpha) 24 and 48 h prior to gamma irradiation were studied using the comet assay and apurinic-apyrimidinic (AP) site radiolabeling. It was demonstrated that the radiation-induced damage assessed by the comet assay in the DNA of peripheral blood lymphocytes (PBLs), splenocytes and thymocytes of mice treated with IFN-alpha before irradiation was considerably less and was repaired more easily in the postirradiation period than that in untreated mice. The DNA of PBLs and splenocytes from interferon-treated mice showed a decrease in the spontaneously occurring and radiation-induced AP sites, as determined immediately and 90 min after irradiation, compared to the level of AP sites in the DNA of untreated mice. The results lead us to assume that IFN-alpha activates the DNA repair systems in the cells of lymphoid tissue.

[Supercopy plasmid based on the replicon from the temperate N15 bacteriophage]. / Sverkhkopiinaia plazmida na osnove replikona umerennogo bakteriofaga N15.

Mini-plasmids, based on the N15 temperate bacteriophage replicon, are described. One of these, N15-203 linear 13.8 kb plasmid, has anomalously high copy number--more than 250 per one bacterial chromosome and the amount of plasmid DNA comprises about half of the total DNA of a cell. This property of N15-203 plasmid is realized only in the strain lysogenic for a N15 phage and is lost for the circular deletion versions of N15-203. The efficiency of transformation of E. coli C (N15) strain is essentially the same for N15-203 and pUC4K plasmids. Insertion of foreign DNA with a size up to 20 kb into BgIII cloning site of N15-203 plasmid does not decrease significantly efficiency of transformation calculated per number of DNA molecules and the total amount of plasmid DNA in a cell. N15-203 plasmid may be used as a vector for molecular cloning of relatively large DNA fragments, and in those biotechnology processes when productivity depends on a vector's copy number.

[Decreased synthetic activity as a possible cause of the death of Escherichia coli bacteria during amino acid starvation]. / Snizhenie sinteticheskoi aktivnosti kak vozmozhnaia prichina gibeli bakterii Escherichia coli vo vremia aminokislotnogo golodaniia.

The work is concerned with studying the breakdown of proteins and RNA when a polyauxotrophic Escherichia coli strain is incubated in a salt solution without amino acids, phosphorus, nitrogen and glucose at 43 degrees C as well as the ability of starving bacterial cells to recommence protein and RNA synthesis (also in the course of phage T4 infection) and to reproduce bacteriophages T4, lambda and MS2. Within the first two hours of the incubation, 12% of proteins and 40% of RNA break down to acid-soluble fragments. Then protein degradation stops while RNA decomposition goes on, but at a lower rate. Within 4-6 h of starvation, the rate of protein and RNA synthesis drops down 4-5 times and the survival rate equals 40-60% when the cells are transferred onto a complete medium. The quantitative characteristics of phages T4, lambda and MS2 reproduction fall down in prestarved cells. The authors speculate that E. coli cells die off in the course of starvation not because some unique structure is destroyed, but owing to the fact that the activity of enzymes and ribosomes gradually declines. As a result, the synthetic activity of the cell drops down abruptly and irreversibly because the enzymes are inactivated and RNA breaks down, which eventually causes cell death.

[Survival of bacteria and the fate of Escherichia coli DNA in amino acid deficiency]. / Gibel' bakterii i sud'ba DNK Escherichia coli pri aminokislotnom golodanii.

The survival rate of an E. coli polyauxotrophous strain AB1157 and the behaviour of its DNA were studied when the strain was incubated for a long time at 43 degrees C in a medium deficient in glucose, phosphates and amino acids. Under these conditions, the survival rate fell down to 10%, but no cell lysis occurred. DNA synthesis stopped within the first two hours of starvation. Neither DNA degradation, despiralization nor decrease of its molecular weight could be detected during the entire starvation. Therefore, the death of E. coli cells under these conditions was not associated with DNA damages.