[Cloning and expression of Vibrio cholerae zonula occludens toxin (zot) gene in Escherichia coli].

Two recombinant plasmids containing the cloned PCR-amplifled Vibrio cholerae zonula occludens toxin (zot) gene was constructed in orientation providing its transcription from lac-promoter. One of them contained also its own zot promoter. The third plasmid was obtained by subcloning a Vibrio cholerae DNA fragment including intact zot and ace (accessory cholera enterotoxin) genes. The expression levels of the cloned genes in Escherichia coli varied depending on a promoter type, host strain and culture conditions. The human intestinal cell line CaCo2 appeared to be a suitable model for assessing the biological activity of toxin preparations. The product of zot gene possessed a marked activity in respect to CaCo2 in spite of the lack of the molecule cleavage and transport of its toxic C-terminal part from alien host cells into the culture media. The constructed recombinant plasmids can be used as a source of molecular hybridization probes; and E.col transformants carrying those plasmids can serve in zot purification both for the scientific and practical purposes.

[Variable nucleotide tandem repeats (VNTR analysis) in Vibrio cholerae 0139 isolated from humans and from water of surface reservoirs in Russia]. / Variabel'nye tandemnye nukleotidnye povtory (VNTR-analiz) u Vibrio cholerae 0139, vydelennykh ot liudei i uz vody poverkhnostnykh vodoemov Rossii.

The comparative study of variable tandem repeats (VNTR analysis) in genomes of V. cholerae 0139 isolated from humans and from water samples taken from surface reservoirs was carried out. The results of the study of the allele state of 5 loci of tandem repeats in 50 strains of vibrios, carried out in the double-primer polymerase chain reaction (PCR), as well as the earlier comparison of the same isolates in the single-primer PCR, showed essential differences and the absence of clonality in the cultures of the clinical and aqueous origin. The suggestion was made that vibrios with individual VNTR genotypes and having no genes ctx and tcpA, isolated from water samples, were epidemic unimportant representatives of the autochthonous microflora of water reservoirs.

[Variable tandem repeat VcA of Vibrio cholerae]. / Variabel'nyi tandemnyi povtor VcA Vibrio cholerae.

Computer analysis revealed seven potential variable-number tandem-repeat (VNTR) loci in the Vibrio cholerae genome. Specific primers were designed to amplify locus VcA located on chromosome 2 and containing a TGCTGT repeat. The locus was found in all tested strains from a V. cholerae strain collection, the repeat number varying 3 to 23. In total, 14 VcA alleles were observed. The VcA locus was proposed as a marker for the molecular typing of V. cholerae strains.

[Evaluation of hypoxic state in experimental animals induced by Yersinia pestis antigens]. / Otsenka gipoksicheskogo sostoianiia u éksperimental'nykh zhivotnykh pod vliianiem antigenov Yersiniia pestis.

Some characteristics of purine metabolism in experimental animals (white mice, clawed jirds and guinea pigs), injected intraperitoneally with Y. pestis "murine" toxin and capsular antigen (Fraction 1), were studied. Under the influence of sublethal doses of these antigens increased levels of guanine and xanthine in blood were noted. Changes in the content of xanthine oxidase in cells were insignificant. In white mice and clawed jirds the activity of succinate dehydrogenase decreased under the action of "murine" toxin and increased after the injection of Fraction 1.

[Increased effectiveness of etiotropic therapy of experimental plague in albino mice at the stage of generalized infection]. / Povyshenie éffectivnosti étiotropnoi terapii éksperimental'noi chymy belykh myshei na stadii generalizatsii infektsii.

Therapeutic efficacies of various drugs were studied comparatively in the treatment of experimental plague in albino mice at the stage of the infection generalization. It was shown that out of the tested drugs such as ciprofloxacin, amikacin, gentamicin, rifampicin and polymyxin B only ciprofloxacin provided a rather high therapeutic effect in the treatment of the plaque septic form. The in vitro and in vivo experiments demonstrated that ciprofloxacin had an antitoxic action on lipopolysaccharide (LPS) and the plague microbe toxin. In comparison to ciprofloxacin, polymyxin B had a higher neutralizing activity. It was found that the efficacy of the experimental plague treatment at the stage of the infection generalization increased with the use of combinations of the drugs with antitoxic and antibacterial activities (ciprofloxacin and polymyxin B).

[The activation of Yersinia pestis "murine" toxin and endotoxin by hemolyzed erythrocytes from mammalian blood]. / Aktivatsiia "myshinogo" toksina i éndotoksina Yersinia pestis gemolizirovannymi éritrotsitami krovi mlekopitaiushchikh.

Y. pestis "mouse" toxin and endotoxin have been found to be capable of being activated with hemolyzed mammalian red blood cells. The LD50 of the activated endotoxin decreases 5-10 times in comparison with the initial preparation. The LD50 of the activated "mouse" toxin decreases 5 times. As revealed in this study, the joint introduction of nonlethal doses of "mouse" toxin and endotoxin is highly toxic for white mice and guinea pigs. The presence of both "mouse" toxin and endotoxin in the toxic mixture is an essential factor for these two species of animals.

[Localization of the DNA segment coding for the synthesis of fraction I on the pYT plague pathogen plasmid]. / Lokalizatsiia otvetstvennogo za sintez fraktsii I uchastka DNK na plazmide pYT chumnogo mikroba.

Fragmentation of the pYT plasmid of the plague pathogen by ten restriction endonucleases has been studied. The evidence has been obtained in support of the possible presence of the movable genetic element containing a HindIII site within the plasmid pYT. The gene encoding the I fraction of the plague pathogen has been cloned. The physical map of the pYT plasmid has been constructed with the use of restriction endonucleases BamHI, XhoI, BstEII, SmaI, EcoRI, and HindIII. The fragment of the plasmid DNA coding for the synthesis of the plague pathogen fraction I has been mapped.

[The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]. / Vliianie 6 MD plazmidy Yersinia pestis EV76 na sostav vneshnemembrannykh belkov Yersinia pseudotuberculosis YPIII.

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.

[Transcription map of a recombinant plasmid carrying a gene for the synthesis of pesticine I and a protein for pesticine I immunity in the plague microbe]. / Transkriptsionnaia karta rekombinantnoi plazmidy, nesushchei geny sinteza pestitsina I i belka immunnosti k pestitsinu I chumnogo mikroba.

Molecules of the plasmids pBR322 and pRD17 have been compared by electron microscopy technique of R-loops visualization. Comparative location of R-loops on the plasmids has been computerized on minicomputer HP9825A due to the program making possible to define the coordinates of the transcription start and direction. The 5.1 kb fragment coding for pesticin I and immunity protein to pesticin I and cloned in pBR322 vector plasmid is flanked by Bam HI-EcoRI sites. Five promoter regions and direction of transcription were localized on the fragment.

[Restriction map of pesticinogenicity plasmid pYP1 of Yersinia pestis]. / Restriktsionnaia karta plazmidy pestitsinogennosti pYP1 Yersinia pestis.

The restriction map of Yersinia pestis pesticinogenicity plasmid pYP1 has been constructed with the use of 18 restriction endonucleases. Plasmid dimensions (6.3 Md) have been specified, the genes for pesticin synthesis, for pesticin immunity protein, fibrinolysin and plasmocoagulase have been localized by molecular cloning of single plasmid DNA fragments in vector plasmid pBR322.

[Comparison of specific recognition sites of adenine and cytosine DNA-methylase of Yersinia Pestis EV 76 C dam and dcm by Escherichia coli methylases]. / Sravnenie spetsifichnosti saitov uznavaniia adeninovoi i tsitozinovoi DNK-metilaz Yersinia pestis EV 76 C dam i dcm metilazami Escherichia coli.

Using enzymatic modelling of in vitro methylation of chromosome DNAs from Yersinia pestis EV 76, E. coli 834 and E. coli C600 RII by DNA methylases of Eco RII and Eco dam as well as of DNA hydrolysis of plasmid pBR 322 from the cells of Y. pestis EV 76, E. coli C600 and E. coli 834 by restrictases of Eco RII and Cfu I, it was found that cytosine DNA methylase from plague bacteria does not correspond to the type of RII methylases of E. coli. Adenyl DNA methylase is related to E. coli methylases type dam and modifies adenine in the nucleotide sequence of GATC.

[Isolation, purification and physicochemical properties of pesticin I from cells of Yersinia pestis strain EV]. / Vydelenie, ochistka i nekotorye fiziko-khimicheskie svoistva pestitsina I iz kletok shtamma EV Yersinia pestis.

Pesticin I has been isolated and purified from Y. pestis strain EV. The homogeneous preparation of Pesticin has been shown to be monomer protein with a molecular weight of 65000 daltons, having three immunologically identical alpha-, beta- and gamma-forms with different isoelectric points. The amino acid composition of Pesticin I is presented. Rabbit anti-serum to the beta-form of the preparation of Pesticin has been obtained.