Non-Specific Porins of Yersinia pseudotuberculosis as Inductors of Experimental Hyperthyroidism in Mice.

In vivo experiments showed that antibodies to OmpC and OmpF porins of Yersinia pseudotuberculosis increased thyroxine (T4) level in the blood of experimental animals. The mice were immunized with different antigens: recombinant OmpF porin in a soluble monomeric form, trimers of OmpC and OmpF porins isolated from the outer membrane, or antibodies to them. The level of thyroxine in the blood of mice immunized with OmpF and OmpC porins increased by 5.47 and 22.3 times, respectively; after immunization with antibodies to these proteins, blood thyroxine increased by 9.28 and 14.29 times. Immunization with recombinant OmpF porin induced no reliable increase in thyroxine level. Hence, the serum to recombinant OmpF porin contains no antibodies specific to conformational antigenic determinants that are present in the protein trimer and, according to our previous findings from molecular docking studies, determine cross-reactions between OmpF porin of Y. pseudotuberculosis and thyroidstimulating hormone receptor.

Expression and purification of a new lectin from mussel Mytilus trossulus.

The gene of mtl from the mussel Mytilus trossulus was cloned into pET-40b(+) expression vector. After expression in E. coli using designed MX-medium an instable soluble form of MTL was obtained. The developed isolation method of the recombinant protein in "semi-denatured" conditions allowed obtaining an active soluble form of the homogenous lectin from the mussel M. trossulus (r-MTL). Both of the lectins had similar antigenic and spatial structures.

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.

Silicon Crystals Formation Using Silicatein-Like Cathepsin of Marine Sponge Latrunculia oparinae.

The cDNA fragment encoding the catalytic domain of the new silicatein-like cathepsin enzyme LoCath was expressed in a strain Top10 of Escherichia coli, extracted and purified via nickel-affinity chromatography. Recombinant enzyme performed silica-polymerizing activity when mixed with water-soluble silica precursor-tetrakis-(2-hydroxyethyl)-orthosilicate. Scanning electron microscopy revealed hexagonal, octahedral and ß-tridimit crystals. Energy dispersion fluorescence X-ray spectrometry analysis showed that all these crystals consist of pure silicon oxide. It is the first report about the ability of marine sponge's cathepsin to polymerize silicon, as well as about the structure and composition of the silicon oxide crystal formed by recombinant cathepsin. Further study of the catalytic activity of silicatein and cathepsin will help to understand the biosilification processes in vivo, and will create basis for biotechnological use of recombinant proteins for silicon polymerization.

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.