[THE EFFECT OF waaL LIGASE GENES DELETION ON MOTILITY AND STRESS ADAPTATION REACTIONS OF YERSINIA ENTEROCOLITICA 6471/76].

The aim of current study was to estimate the influence of waaL(OS) and waaL(PS) genes deletion on lipopolysaccharide (LPS) synthesis, bacterial motility and stress resistance of bacteria Yersinia enterocolitica 6471/76. Single and double waaL mutants were created by replacing the wild-type alleles in bacterial chromosome for mutant ones. The phenotypes of mutants were visualized by DOC-PAGE gels stained with silver and immunoblot with specific to O-polysaccharide and outer core monoclonal antibodies. Bacterial motility was evaluated by the diameter of the migration zone. Wild type bacteria and mutants were analyzed by bacterial growth curves in a hypertonic medium. Participation of WaaL ligases in resistance to osmotic pressure was found only in case of both ligese genes deletion. Also the YeO3_os_ps mutants showed motility decreasing, which recovered after adding a functionally active gene. Thus, deletion of both waaL ligase genes lead to a drastic reduction in bacterial motility and increase their sensitivity to hypertonic medium that can indirectly characterize biological role of WaaL ligases.

[Rat immunoreactivity in experimental modeling of acute autohemorrhagic bihemispheric stroke in the presence of staphylococcal infection].

Experimental studies were made of rat immunoreactivity in experimental modeling of acute hemorrhagic stroke in staphylococcal infection. Bactericidal activity and reserve bactericidal activity of neutrophils in NBT-test enhance while mid-molecular CIC level decreases because of phagocyte activation. ABA high titers in blood reflect activation of autoimmune processes. Splenic mass increased suggesting activation of proliferation of splenic lymphoid cells or enhanced migration of immature lymphoid cells from bone marrow to the spleen. Experimental animals had suppressed functional activity of main lymphoid cells populations in blast-transformation reaction which may affect the course of early recovery, promote activation of infectious bacterial process, autoaggression. The results of the studies can serve the basis for further investigations of immune mechanisms involved in development of hemorrhagic stroke in the presence of infectious process.

[The characteristics of the polyclonal action and immunomodulating activity of bacterial peptidoglycans in oppositely reacting mouse strains]. / Osobennosti poliklonal'nogo deistviia i immunomoduliruiushchei aktivnosti bakterial'nykh peptidoglikanov u oppozitno reagiruiushchikh linii myshei.

The data on the specific features of the polyclonal action of gram-negative bacteria on primary immune response to sheep red blood cells in oppositely reacting mouse strains are presented. Staphylococcus aureus and Bacterium flavum peptidoglycans have been found to increase the amount of antibody producing cells to thymus-dependent antigen only in the spleen of low-responsive mice. At the same time differences in the amino acid composition of these heteropolymers do not affect their stimulating activity. Differences between the capacity of B. flavum peptidoglycans to induce the activation of B cells in mice of different strains have been established.

[The effect of the structural components of Staphylococcus aureus on the interaction of T- and B-lymphocytes]. / Vliianie strukturnykh komponentov Staphylococcus aureus na vzaimodeistvie T- i B-limfotsitov.

The results of studies on the influence of staphylococcal peptidoglycan and cell walls on the cooperative interaction of T- and B-lymphocytes in the process of immune response to thymus-dependent antigen are presented. Peptidoglycan has been found to produce, depending on its dose, a suppressive and stimulating effect on the interaction of T- and B-cells. Cell walls exhibit only stimulating action under the same experimental conditions. The suppressive action of peptidoglycan is mediated by T-lymphocytes.

[Induction of delayed hypersensitivity in guinea pigs by staphylococcal cell wall components]. / Induktsiia giperchuvstvitel'nosti zamedlennogo tipa u morskikh svinok komponentami kletochnoi stenik stafilokokka.

The subcutaneous injection of 100 micrograms of staphylococcal cell-wall peptidoglycan in Freund's incomplete adjuvant leads to the development of sensitization which can be detected on days 7-35 by the splenocyte migration inhibition test. Sensitization can be detected with peptidoglycan, as well as with staphylococcal cell walls. Teichoic acids produce no such effect. The sensitization of guinea pigs with whole staphylococcal cells introduced in three injections also leads to the development of delayed hypersensitivity specific to staphylococcal cell walls, peptidoglycan, but not teichoic acids.

[The effect of staphylococcal peptidoglycan on macrophage interferon formation and bactericidal activity]. / Vliianie stafilokokkovogo peptidoglikana na interferonoobrazovanie i bakteritsidnuiu aktivnost' makrofagov.

Staphylococcus aureus cell-wall peptidoglycan has been shown to stimulate the bactericidal activity of peritoneal exudate macrophages. A more pronounced activating effect of peptidoglycan on the bactericidal activity of macrophages has been detected in the stimulated test. Peptidoglycan in doses of 5-100 mg induced interferon production by intact mouse splenocytes after 24-hour exposition with it. Peptidoglycan has been found to produce the most intensive effect on interferon production when injected intraperitoneally in a dose of 100 mg after 4-hour exposition.

[Effect of the cell components of Staphylococcus aureus on the functional activity of hemopoietic cells in mice]. / Vliianie komponentov kletok Staphylococcus aureus na funktsional'nuiu aktivnost' gemopoéticheskikh kletok myshei.

The data on the stimulating action of S. aureus cells, strains B-243, 2287, Wood-46, Cowan I, as well as cell-wall peptidoglycan, on the formation of endogenous colonies in the spleen of sublethally irradiated mice are presented. Teichoic acid, S. aureus ribosomal and cytoplasmic antigens produced no such effect. Whole S. aureus cells and their components were incapable of activating transitory colonies in the spleen of sublethally irradiated mice. After immunization with cell walls, peptidoglycan and protein A the mice showed a rise in the absolute and relative content of blood-forming stem cells in the marrow and the spleen. Killed S. aureus cells increased the relative content of blood-forming stem cells in the marrow, while in the spleen a rise in both absolute and relative content of such cells occurred, which was detected in the exocolonization test.

[Effect of staphylococcal substance on the phagocytic activity of mouse peritoneal exudate cells]. / Vliianie substantsii stafilokokka na fagotsitarnuiu aktivnost' kletok peritoneal'nogo ékssudata myshei.

The cell walls, peptidoglycan and whole cells of St. aureus inhibit the phagocytosis of sheep red blood cells in the culture of mouse peritoneal macrophages. The intraperitoneal injection of cell-wall components (peptidoglycan, teichoic acids) and whole staphylococcal cells stimulates phagocytosis at different periods after immunization, then follows suppression. Teichoic acids seem to act as agents suppressing phagocytic activity during 24 hours after immunization.

[Mitogenic activity of the antigens isolated from different Staphylococcus aureus strains in relation to mouse and guinea pig splenocytes]. / Mitogennaia aktivnost' antigenov, vydelennykh iz raznykh shtammov zolotistogo stafilokokka, v otnoshenii splenotsitov myshei i morskikh svinok.

The mitogenic effect of corpuscular antigens with respect to the splenocytes of animals was found to depend on the strain of Staphylococcus aureus. The maximum synthesis of DNA in the cells was induced by corpuscular antigen Smith and the minimum synthesis, by Wood-46. The synthesis of DNA was activated in both B- and T-splenocytes in response to corpuscular antigens Wood-46, Cowan-1 and Smith, as well as to the cell wall and protein A. Peptidoglycan produced a mitogenic effect only in B-lymphocytes, and teichoic acid showed no mitogenic activity in mouse splenocytes. The mitogenic effect of staphylococcal antigens on splenocytes depended on the dose of the antigen and the time of cultivation. After 48-hour cultivation the incorporation of 3H-thymidine into the DNA of mouse cells was 5 times higher than into the DNA of guinea-pig cells. The optimum mitogenic dose in thymectomized BALB/c mice with respect to splenocytes was higher than in normal BALB/c mice practically by one order.

[Heterogeneity of peptidoglycans of bacterial origin]. / Issledovanie geterogennosti peptidoglikanov bakterial'nogo proiskhozhdeniia.

Heterogeneity of peptidoglycans of Staphylococcus aureus of strains 3272 and Wood-46, Brevibacterium flavum, Micrococcus lysodeikticus has been studied by the method of electrophoresis in the system polyacrylamide gel--sodium dodecylsulfate. It is shown that peptidoglycans of different strains of S. aureus and other species of gram-positive bacteria treated with ultrasound are a complex of fractions with 3 basic high-molecular components with M 100, 92 and 84 kDa, while two main fractions with Mm 92 and 84 kDa are identified in peptidoglycans drugs treated with ultrasound.

[Effect of staphylococcal peptidoglycan on the activity of Ca2+-ATP-ase from the sarcoplasmic reticulum in the early stages of ionizing radiation effect]. / Vplyv stafilokokovoho peptydohlikany na aktyvnist' Ca2+-ATP-azi sarkoplazmatychnoho retykulumu hnoho rannikh etapakh promenevoï diï.

It is established that peptidoglycan of Staphylococcus aureus possess superficial activity and can render influence on the functional activity of biomembranes. It tends to increase Ca(2+)-ATP-ase activity in the sarcoplasmic reticulum membranes, but no changes have been observed in case of the enzyme solubilization. The effect of peptidoglycan was more expressed, if it was used in the early period of acute radiation injury (1 and 24 h) in a dose of 0.21 Cl/kg for samples with irradiation-induced decrease of Ca(2+)-ATP-ase activity.

[Changes in the intensity of DNA synthesis in immunocompetent mouse tissues by peptidoglycans of bacterial origin]. / Zminy intensyvnosty syntezy DNK u imunokompetentnykh tkanynakh mish peptidohlikanamy bakteriial'noho pokhodzhennia.

The data are presented on the effect of activation of by peptidoglycans from cell walls of Staphylococcus aureus and Brevibacterium flavum on the DNA synthesis intensity in the mice spleen, thymus and bone marrow in vivo. Maximum of the peptidoglycan influence on the DNA synthesis intensity is observed 12-36 hours after injection. As a rule, higher doses of the studied peptidoglycans (1 mg per one animal) activate the DNA synthesis more efficiently. Peptidoglycan from Br. flavum stimulates the DNA synthesis in all tissues more intensively then the peptidoglycan from S. aureus, which may be a result of its monomers' structure.

[DNA synthesis in lymphocytes of irradiated animals as affected by mitogens and Staphylococcal antigens]. / Syntez DNK u limfotsytakh oprominenykh tvaryn pid vplyvom mitoheniv i antyheniv stafilokoka.

Proliferation activity of lymphocytes from rats was studied using micromethod of blast transformation reaction after exposure of X-ray irradiation (absorbed doses for animals are 0.5 and 1 Gr). The reducing of the functional activity of lymphocytes was found. There was difference in lymphocyte response induced by standard mitogens (PHA, ConA, LPS) and antigens of Staphylococcus protein A and peptidoglycan. The irradiation protective effects of Staphylococcus substances were found.

[The reaction of anterior hypothalamic nuclei and of the endocrine glands to the administration of staphylococcal peptidoglycan]. / Reaktsiia iader perednego gipotalamusa i éndokrinnykh zhelez na vvedenie stafilokokkovogo peptidoglikana.

The results of investigation of the influence of Staphylococcus peptidoglycan on supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei, adrenal and thyroid glands of CBA mice was studied. Alterations in the SON and PVN secretory cells were found. The percentage of neurocytes that actively synthesize and secrete the Gomori [correction of Homory]-positive substance increased. Biosynthetic functions and secretion of the hormone was enhanced. In adrenal glands, alterations were found only in the bundled zone.

[The diagnostic importance of antibodies to the cell wall peptidoglycan of Staphylococcus in the blood serum of children with a staphylococcal infection]. / Diagnosticheskoe znachenie antitel k peptidoglikanu kletochnoi stenki stafilokokka v syvorotke krovi detei so stafilokokkovoi infektsiei.

High titers of antibodies to peptidoglycan of staphylococcus aureus were found in children with purulent-inflammatory diseases of staphylococcal etiology. This may be used for the diagnosis of staphylococcal infection.

[The development of a coagglutination reaction using staphylococcal peptidoglycan for the diagnosis of staphylococcal infection]. / Razrobka reaktsiï koahliutynatsiï z vykorystanniam stafilokokovoho peptydohlikanu dlia diahnostyky stafilokokovoï infektsiï.

Our objective in this study was to develop and assess the diagnostic value of a coagglutination test with making use of peptidoglycane of Staphylococcus aureus in the identification of diseases of staphylococcal etiology. A total of 166 patients with diseases of staphylococcal etiology were examined. A test was elaborated of coagglutination with making use of peptidoglycane of Staphylococcus aureus for a differential identification of antibodies to peptidoglycan in healthy persons and patients with staphylococcosis.

[Optimization of recombinant clumping factor of Staphylococcus aureus expression in Escherichia coli XLI-BLUE]. / Optymizatsiia ekspresiï rekombinantnoho plastivtsevoutvoriuiuchoho faktora Staphylococcus aureus u klitynakh Escherichia coli XLI-BLUE.

Experiments for optimization of the clfA gene expression in E. coli XLI-Blue cells were conducted. The reducing of the strain efficiency and the elimination of the plasmid during the cultivation of a producer have been shown. Several methods for raising the amount of plasmid-containing cells and increasing the yield of recombinant protein were proposed. Bacterial growth medium was selected to make this process more efficient. In such conditions after IPTG induction of the recombinant cells we have received high level of a soluble protein (about 30% of total cellular protein). The recombinant protein containing the His tag on N-terminus was purified on IDA-Sepharose column with high yield.

[Stimulation of DNA synthesis in guinea pig spleen lymphocytes by staphylococcal peptidoglycan]. / Stimuliatsiia sinteza DNK v limfotsitakh selezenki morskikh svinok peptidoglikanom stafilokokka.

The mode of action of peptidoglycan on DNA synthesis in spleen lymphocytes of intact guinea-pigs was studied. It was found that peptidoglycan isolated from Staphylococcus aureus 2287 is mainly mitogenic for B lymphocytes that have IgG receptors on their surface. In contrast to lipopolysaccharide from Salmonella typhimurium, it seems that peptidoglycan is also mitogenic for less differentiated B cells.