Optimization of cold-adapted alpha-galactosidase expression in Escherichia coli.

α-Galactosidase (α-PsGal) of the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701 was cloned into the pET-40b(+) vector to study its properties and to develop an effective method for modifying human B-erythrocytes into O-blood group. The use of heat-shock as a pre-induction treatment, IPTG concentration of 0.2 mM and post-induction cultivation at 18 °C for 20 h in the developed MX-medium allowed increasing the recombinant Escherichia coli Rosetta (DE3)/40Gal strain productivity up to 30 times and the total soluble α-PsGal yield up to 40 times.

[BIOLOGICAL ACTIVITY OF YERSINIA PSEUDOTUBERCULOSIS TOXINS].

AIM: Study of effect of heat-labile (HLT) and thermostable (HST) lethal toxins of Yersinia pseudotuberculosis on the development of embryos of sea urchin Strongylocentrotus intermedius, processes of biosynthesis of nucleic acids and protein in embryo cells and activity of nucleoside- kinases of sea urchin. Materials-and methods. Y pseudotuberculosis strains 2517 (pYV-) and 512 (pYV48MD, pYV82MD) were used for isolation of HLT and HST Gametes and embryos of sea urchin S. intermediuswere used to carry out the experiments and isolate nucleoside-kinases. RESULTS: , Both of the studied toxins of Y pseudotuberculosis possessed, spermiotoxic effect and reduced fertilizing ability of sea urchin spermies. HLT LD50 was 1 µg/ml, and HST - 2 µg/ml. Toxins affected the development of embryos of sea urchin resulting in severe morphologic damages, cessation ofthe development of embryos at early stages of embryogenesis, destruction of cells and death of embryos. Wherein; damaging effect of HLT was observed at lower concentrations compared with HST HLT inhibited DNA and RNA biosynthesis at concentrations of 1-2 µg/ml. HST did not affect biosynthesis of nucleic acids even at high concentrations, but inhibited protein biosynthesis in sea urchin embryos. HLT did not reduce the level of inclusion of labeled amino acids into embryo cells. HLT had inhibiting effect on the activity of thymidine- and uridine-kinase of sea urchin, whereas HST did not affect these enzymes. CONCLUSION: Both of Y pseudotuberculosis protein toxins affect the development of sea urchin embryos, however, mechanisms of action of HLT and HST on embryos and processes occurring in them differ.

[CHARACTERISTICS OF FORMATION, INHIBITION AND DESTRUCTION OF YERSINIA PSEUDOTUBERCULOSIS BIOFILMS FORMING ON ABIOTIC SURFACES].

AIM: Detection of conditions of Yersinia pseudotuberculosis biofilm formation, their quantitative testing. MATERIALS AND METHODS: Y. pseudotuberculosis strains, nutrient media, standard 96-well polystyrene plates, crystal violet dye as well as bacteriologic, spectrophotometric, statistical methods were used. RESULTS: All the studied Y pseudotuberculosis strains formed a well expressed biofilm on abiotic surface during cultivation of bacteria in 200 µl of a plate well at a temperature of 20-22°C for 4-7 days. Bacteria CFU number in biofilm reduced by day 10 of incubation. DNAse I was found to inhibit biofilm formation, and also partially destroyed mature Y. pseudotuberculosis biofilm. The presence of DNA in extra-cellular matrix of biofilm was shown. CONCLUSION: An ability of Y. pseudotuberculosis to form biofilm on abiotic surface was established. The conditions of biofilm formation were determined. Inhibiting effect of DNAse I on Y. pseudotuberculosis was shown.

[Regulation of Yersinia pseudotuberculosis major porin expression in response to antibiotic stress].

The OmpF porin gene expression in Yersinia pseudotuberculosis in response to antibiotics of two different classes (kanamycin and nalidixic acid) was analyzed using quantitative PCR and a fluorescence reporter system. Both antibiotics downregulated the expression of the ompF gene. The nalidixic acid significantly reduced ompF expression, while kanamycin, for which porins are considered to be an alternative transport route, only slightly reduced the ompF level.

Detection and genotyping of Helicobacter pylori gene vacA in children with gastroduodenal diseases and in adults with gastric cancer in Vladivostok.

Geographical distribution of individual genotypes of Helicobacter pylori, predominance of virulent types in various regions of Russia, particularly in the Prymorye Territory, remains unclear. We examined 115 children with various gastroduodenal pathology and 33 patients with gastric cancer, of which 57.39 and 60% respectively were infected with H. pylori. All samples positive for H. pylori were further analyzed for gene vacA mosaicism. In all clinical subgroups, variants s1 and m1 predominated; the frequency of genotype s1 was significantly increased (1.3-fold) in the group of cancer patients in comparison with the group of children with gastroduodenal pathology. Three variants of allele combination of signaling and middle regions of the vacA gene (s1m1, s1m2, and s2m2) were revealed; s1m1 was the most frequent in both groups. We suggest that this genotype is a marker of complicated course of gastroduodenitis and a factor of gastric cancer development in local population.

Catalytic properties and amino acid sequence of endo-1→3-ß-D-glucanase from the marine mollusk Tapes literata.

A specific 1→3-ß-D-glucanase with molecular mass 37 kDa was isolated in homogeneous state from crystalline style of the commercial marine mollusk Tapes literata. It exhibits maximal activity within the pH range from 4.5 to 7.5 at 45°C. The 1→3-ß-D-glucanase catalyzes hydrolysis of ß-1→3 bonds in glucans as an endoenzyme with retention of bond configuration, and it has transglycosylating activity. The K(m) for hydrolysis of laminaran is 0.25 mg/ml. The enzyme is classified as a glucan endo-(1→3)-ß-D-glucosidase (EC 3.2.1.39). The cDNA encoding this 1→3-ß-D-glucanase from T. literata was sequenced, and the amino acid sequence of the enzyme was determined. The endo-1→3-ß-D-glucanase from T. literata was assigned to the 16th structural family (GHF 16) of O-glycoside hydrolases.

[Ribonuclease from the hepatopancreas of the red king crab Paralithodes camtschatica].

Three enzyme preparations, two acid and one alkaline RNases, were isolated from the hepatopancreas of the red king crab Paralithodes camtschatica using DEAE-cellulose chromatography and gel chromatography. The alkaline RNase was activated by Mg2+ ions and had a pH optimum of 7.2; the acid RNases, a pH optimum of 5.5. The molecular weight of the alkaline RNase was 19 kDa; two acid RNases, 33 and 70 kDa, respectively. The enzymes exhibited a sufficiently high thermostability (IT50 = 53-55 degrees C) and were strongly inhibited by NaCl (IC50, 0.1-0.25 M). The alkaline RNase exhibited no specificity for heterocyclic bases, whereas the acid RNases hydrolyzed poly(U) and poly(A) at maximum rates.

[Development of a multiplex PCR for detection of the Yersinia genus with identification of pathogenic species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica)].

To identify the Yersinia genus and pathogenic species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) in a single reaction a multiplex PCR technique was developed. It was optimized by five compounds of PCR buffer and temperature of primers annealing. The multiplex PCR provides an improved and rapid method for detection of the Yersinia genus and identification of pathogenic species.

[Thymidine and thymidylate kinases from the scallop Mizuhopecten yessoensis gonads].

Thymidine and thymidylate kinases were isolated from the gonads of scallop Mizuhopecten yessoensis. The enzymes were purified 537- and 100-fold, respectively, and were free of phosphatase and ATPase impurities. Ions of bivalent metals and ATP were necessary for both the nucleoside and nucleotide kinase activities; the pH optimum fall into the range of 7.5-8.5. KCl and NaCl at a concentration of up to 100 mM had no inhibiting effect on the activities of these scallop enzymes. Thymidine kinase catalyzed thymidine, and, at a lower rate, deoxycytidine phosphorylations did not utilize ribo- and deoxyribonucleosides, as well as pyrimidine ribonucleosides, as a phosphate acceptor. Thymidylate kinase phosphorylated TMP and dCMP with an efficiency of about 30%. In addition to ATP, these enzymes can also utilize with different efficiencies dATP, dGTP, GTP, UTP, and CTP as a donor of phosphate groups. Thymidine kinase activity was inhibited by TMP, TTP, and dCTP.

[Phosphatases and phosphodiesterases isolated from the red king crab (Paralithodes camtschatica) hepatopancreas].

Five enzymes have been isolated from the hepatopancreas of the red king crab Paralithodes camtschatica by means of ion exchange and gel chromatography: two acid (AcP) and one alkaline (AlkP) phosphmonoesterases, one alkaline phosphodiesterase (AlkPD), and one acid phosphodiesterase (AcPD). The pH optimum values of these enzymes are: AlkPs and AlkPD, 7.5; AcP, 5.5; and AcPD, 5.0. The activity of AlkP and AlkPD demands Mg2+ ions. The molecular weights of the enzymes (kDa) are the following: AlkP, 80: AcPs, 80 and 82; AlkPD, 51; and AcPD, 57. The enzymes are relatively thermostable (ID50 from 47 to 62 degrees C). AlkP is inhibited by NaCl (IC50 at 0.4 M). The AcP, AcPD, and AlkPD activities are tolerant of high ionic strength.

[Influence of thermolabile lethal toxin of Yersinia pseudotuberculosis on development of sea urchin embryos and biosynthesis of DNA and RNA in embryonic cells].

Influence of thermolabile lethal toxin of Y. pseudotuberculosis on the development of embryos of sea urchin (Strongylocentrotus intermedius) and on biosynthesis of nucleic acids in embryonic cells was studied. Thermolabile lethal toxin affected metabolic processes of cells by inhibiting DNA and RNA synthesis. It had damaging action on developing embryos of sea urchin causing morphological changes and, as a consequence, death of embryos.

Estrogenic activity of triterpene glycosides in yeast two-hybrid assay.

Estrogenic potency of six triterpene glycosides, Holothurin A, Holotoxin A1, Frondoside A, Cucumarioside A2-2 and Cauloside C, that are natural products and semi-synthesized Ginsenoside-Rh2, were examined with yeast two-hybrid system, including expressed genes of human estrogen receptor, hERalpha, the co-activator TIF2 and lacZ as a reporter gene. Only Ginsenoside-Rh2 exhibited significant moderate estrogenic activity in the concentration range of 10(-7) to 10(-6)M. Its effect was approximately 30% of the activity of 17beta-estradiol applied at half-effective concentration. This indicates Ginsenosides-Rh2 is a weak phytoestrogen. The sea cucumber triterpene glycosides, Holothurin A, Holotoxin A1, Cucumarioside A2-2 and Frondoside A, and plant glycoside Cauloside C had no appreciable estrogenic activity. Data obtained by yeast two-hybrid assay reflect structure-activity relationship between tested compounds and 17beta-estradiol. Only Ginsenoside-Rh2 has some similarity in chemical structure with 17beta-estradiol that might explain affinity of this glycoside to the hERalpha receptor.

A highly active alkaline phosphatase from the marine bacterium cobetia.

An alkaline phosphatase with unusually high specific activity has been found to be produced by the marine bacterium Cobetia marina (strain KMM MC-296) isolated from coelomic liquid of the mussel Crenomytilus grayanus. The properties of enzyme, such as a very high specific activity (15000 DE U/1 mg of protein), no activation with divalent cations, resistance to high concentrations of inorganic phosphorus, as well as substrate specificity toward 5' nucleotides suggest that the enzyme falls in an intermediate position between unspecific alkaline phosphatases (EC 3.1.3.1) and 5' nucleotidases (EC 3.1.3.5).

[Primary structures of actinoporins from sea anemone Oulactis orientalis].

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with specific primers to the N-terminal regions of the 0. orientalis actinoporin sequences and to the C-terminal region, which is known to be highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were established by sequencing the cDNA clones obtained by the fast amplification of 3'-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain lower hemolytic activities of Or-A and Or-G than that of actinoporins from the tropical species.

[Yersinia pseudotuberculosis nucleoside-kinase].

Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.

Recombinant α-NAcetylgalactosaminidase from Marine Bacterium-Modifying A Erythrocyte Antigens.

A plasmid based on pET-40b was constructed to synthesize recombinant α-N-acetylgalactosaminidase of the marine bacterium Arenibacter latericius KMM 426T (α-AlNaGal) in Escherichia coli cells. The yield of α-Al- NaGal attains 10 mg/ml with activity of 49.7 ± 1.3 U at 16°C, concentration of inductor 2 mM, and cultivation for 12 h. Techniques such as anion exchange, metal affinity and gel filtration chromatography to purify α-AlNaGal were applied. α-AlNaGal is a homodimer with a molecular weight of 164 kDa. This enzyme is stable at up to 50°C with a temperature range optimum activity of 20-37°C. Furthermore, its activity is independent of the presence of metal ions in the incubation medium. 1H NMR spectroscopy revealed that α-AlNaGal catalyzes the hydrolysis of the O-glycosidic bond with retention of anomeric stereochemistry and possesses a mechanism of action identical to that of other glycoside hydrolases of the 109 family. α-AlNaGal reduces the serological activity of A erythrocytes at pH 7.3. This property of α-AlNaGal can potentially be used for enzymatic conversion of A and AB erythrocytes to blood group O erythrocytes.

Some properties of alkaline DNases of tentacles of actinia Radianthus macrodactylus and their hemolytic activity.

Two alkaline DNases of tentacles of actinia Radianthus macrodactylus, referred to as alk DNase I and alk DNase II, respectively, have been purified up to apparent homogeneity with consecutive column ion exchange chromatography and gel filtration. Both enzymes have a lot of common properties, such as the ability to hydrolyze very effectively p-nitrophenyl-5'-TMP and heat-denatured DNA. They both have no preferential specificity to the sugar component of the nucleic acids and effectively digest ribopolymers. Their ability to hydrolyze supercoiled DNA of the pBR322 plasmid and linear DNA of the lambda phage by "miscellaneous" exo- and endonucleolytic types of attack and to produce nucleosides, nucleotides and short oligonucleotides suggests their similarity with phosphodiesterase I (5'-exonuclease, oligonucleate 5'-nucleotidohydrolase; E.C. 3.1.4.1), isolated from rattle snake Crotalus adamenteus venom. Alk DNase II has been revealed to have some uncommon properties, such as phosphomonoesterase and hemolytic activities. The protein causes a very potent lysis of human and rabbit erythrocytes. The ability of alk DNase II to precipitate some components of normal human and rabbit blood serum as well as the inhibition of this reaction by fucose but not by another monosaccharides suggest the enzyme to have a lectin-like activity. The appearance of only one protein band during electrophoresis of alk DNase II in denaturation conditions suggests that all activities are inherent to the same molecule of protein. The possible role of alkaline DNases in the toxic effect of burning by actinia tentacles is discussed.

[Site of nuclear DNA replication in embryonic cells of the sea urchin Strongylocentrotus intermedius]. / Issledovanie mesta replikatsii iadernoi DNK e embrinov morskogo ezha Strongylocentrotus intermedius.

In the sea urchin embryonic cells, all newly synthesized nuclear DNA (n-DNA) pulse-labeled by 3H-thymidine was found within DNA-membrane complex (DNA-mc) isolated by centrifugation of lysates of nuclei after their treatment with Sarkosyl, Brij-35, or sodium dodecylsulfate through neutral sucrose (10--30%) gradients. This attachment has been shown not to be an artifact due to the unspecific effect of the detergents or the destabilization of the secondary structure of n-DNA because the association of the exogenous 14C-DNA with nuclear membrane and chromatin did not occur during the isolation of the DNA-mc. n-DNA was not replaced from DNA-mc when the latter was isolated in the excess of unlabeled denatured DNA. n-DNA associated with DNA-mc behaved as a precursor of chromosomal DNA. It is suggested that in sea urchin embryonic cells the synthesis of nuclear DNA is carried out by the replicative complex attached to the nuclear membrane.

[Mechanism of ATP-dependent DNAse purified from sea urchin Strongylocentrotus intermedius embryos]. / Issledovanie mekhanizma deistbiia ATP-zavisimoi DNKazy iz émbrionov morskogo ezha Strongylocentrotus intermedius. I. Deistvie na kol'tsevye DNK.

Supercoiled DNAs of the SV40 virus and phi X174 phage have been studied under the effect of ATP-dependent DNase purified from sea urchin embryos. The studies showed a relaxing activity of the enzyme in relation to the supercoiled DNA. The supercoiled DNA treated with the enzyme in the absence of ATP was ultracentrifuged in a linear (5-20%) alkali sucrose gradient and the DNA preparations obtained by Davis formamide technique were examined by electron microscopy to demonstrate accumulation of double stranded DNA (RFIII). After addition of ATP to the incubation mixture only relaxed (RFII) and supercoiled (RFI) molecules of circular DNAs were observed among the products of enzyme hydrolysis of supercoiled DNAs.

[Various characteristics of Yersinia pseudotuberculosis determined by plasmid 45 Md associated with calcium dependence]. / Nekotorye osobennosti Yersinia pseudotuberculosis, opredeliaemye plazmidoi 45 Md, sviazannoi s kal'tsiizavisimost'iu.

The clones of two types (T+ and T-) have been identified among the strains of Y. pseudotuberculosis. The difference in the morphology of the clones is based on the ability of T+ clones to agglutinate in the process of growth. The identified clones are different in calcium dependence, in the ability to agglutinate and in their virulence for the laboratory animals. The differences have been proved to be connected with the presence of a 45 Md plasmid in T+ cells. Replication of this plasmid is suppressed by the acridine orange dye in concentrations of 12.5 or 25.0 mkg X ml-1. The plasmid is spontaneously lost from the strains during their continuous storage. The microscopy of colonies permits the selection of clones with the parental phenotype from the populations having lost the 45 Md plasmid.