Proline-dependent wild strains and proline-dependent mutants of the plague microbe]

It is found that the growth of Yersinia pestis wild strains, isolated from Citellus musicus Menetrié in the Central Caucasus, depends on the presence of proline in the medium. Proline can not be substituted by glutamic acid, other amino acids or vitamins. 28 proline-requiring mutants were selected from Y. pestis marmot strain 20b. Three groups of proline-requiring Y. pestis mutants are established, similar to those of Escherichia coli. The requirement of proline does not affect the virulency, pigment formation and calcium dependence.     

Antibiotic sensitivity of plague microbe strains from foreign countries]

The method of serial dilutions on the Hottinger agar was applied to comparative assay of antibiotic sensitivity in 50 strains of the plague microbe isolated abroad and in 5 strains isolated in the plague focus in the Central Caucasus. The antibiotics used in the assay were the following: streptomycin, gentamicin, doxycycline, monomycin, kanamycin, tetracycline, erythromycin, ristomycin, lincomycin and polymyxin M. Irrespective of the origin, all the isolates were resistant to erythromycin, lincomycin and polymyxin M. The levels of the sensitivity to the other antibiotics were different. The data serve as a ground for the statement that there is no tendency to development of antibiotic resistance in the plague microbe in patients treated with high doses of the antibiotics and mainly streptomycin. Along with streptomycin, such antibiotics as gentamicin, tetracycline, doxycycline and kanamycin are useful in the therapy of plague and require further investigation.     

Response, to polymyxin, of plague microbe strains isolated from various natural foci and of their mutants with a decreased requirement in nutritional factors

Investigation of the response to polymyxin of 65 strains of Pasteurella isolated from various foci and 46 their back mutants showed that all of them were usually highly resistant to polymyxin (MIC 200--500 micrograms/ml). The Pasteurella strains isolated in the Gissaro-Darvaz natural focus, Turkey and Congo were highly sensitive to polymyxin (MIC 10--25 micrograms/ml). Single cultures highly sensitive to the antibiotic were detected among the polymyxin-resistant strains. Polymyxin-sensitive mutants of these cultures with lowered requirements in the growth factors were obtained.     

Isolation and properties of several auxotrophic mutants of a highly virulent strain of the plague microbe]

2432 stable auxotrophic mutants were selected from high virulent Yersinia pestis strain 20b after treatment with nitroso guanidine. They were deficient in amino acids (arginine, aspartic acid, citrulline, glycine, glutamic acid, histidine, isoleucine, serine, leucine, lysine, ornithine, proline, tryptophan, tyrosine, valiney, pyrimidine and vitamins (riboflavin, thyamine, nicotinamide). Some mutants were two- and three-fold dependent. The leucine-, histidine-, purine-dependent mutants were isolated with the high frequency. All the mutants, like their original strain, grew in R-form; they were sensitive to diagnostic phages, had pesticine-fibrinolysin-coagulase sustem (fraction I) and were calcium-dependent. P+ cultures of auxotrophs were not virulent for laboratory animals.     

Spheroplasts of plague microbe strains from the Transcaucasian uplands and their capacity for pesticin synthesis]

Spherical formations of the plague microbe strains from the Transcaucasian Upland, I plague microbe strain of the sandwort variety and I strain of the marmot variety were obtained under the effect of lithium chloride. They had the remains of the cell wall, were viable, sensitive to osmotic shock, preserved sensitivity to the specific bacteriophage and pesticins. All this was evident of isolation of the spheroplasts of the plague microbe. The spheroplasts showed a capacity for pesticin production. The pesticin synthesis by the spheroplasts of the plague causative agent from the Transcaucasian Upland increased with an increase in the content of lithium chloride in the medium. The largest inhibiiton zones were observed, when 0.7-0.8 per cent of lithium chloride were present in the medium. In the spheroplasts of the plague causative agent from the Mountain Altai (the marmot variety) the pesticin synthesis was inhibited with an increase in the content of lithium chloride in the medium. The activity spectrum of the pesticins of the spheroplasts of the plague causative agent from the Transcaucasian Upland and the spheroplasts of the strains of the marmot and sandwort varieties was broader than that of the rod-like forms of these strains. The indicator properties were found in the strains of the plague microbe of the marmot and sandwort varieties with respect to the pesticins of the spheroplasts of the sel-like producing organisms and organisms from the Transcaucasian Upland.     

Isepamycin in prophylaxis and treatment of experimental plague due to FI+ and FI- variants of plague microbe]

The efficacy of isepamycin vs. other aminoglycosides was studied in vitro and on albino mice with experimental plague due to natural antigen valuable strains of the plague microbe and the pathogen variants deprived of the ability to produce the capsular antigen fraction I (FI- phenotype). The MICs of isepamycin for the strains of the plague microbe (20 FI+ and 20FI-) were 1.0-4.0 mg\l, that did not differ from those of streptomycin, kanamycin, amikacin and tobramycin. The ED50 of isepamycin in the prophylaxis and treatment of the experimental plague of the mice had no statistically significant differences from the ED50 of the other aminoglycosides. The efficacy index of isepamycin was > 10(4), that did not differ from that of streptomycin, amikacin and gentamicin, irrespective of the strain phenotype (Y. pestis 231 FI+ or Y. pestis 231 FI-). The same as the other aminoglycosides, isepamycin in doses equivalent to the human average daily doses, protected 80-100% of the albino mice from death when used in the prophylaxis and therapy of plague irrespective of the strain phenotype. The results of the study made it possible to consider isepamycin as an agent promising for the prophylaxis and treatment of plague.     

Formation of virulent antigen-modified mutants (Fra-, Fra-Tox-) of plague bacteria resistant to rifampicin and quinolones]

Experiments were performed with two strains of plague bacteria--231 (isolated from marmot) and 358 (isolated from human) and their isogenic variants with Fra- and Fra-Tox- phenotype. Mutants resistant to rifampicin (Rifr) and nalidixic acid (Nalr) appeared independently of pathogen phenotype and genotype with frequency n.10(-8)-n.10(-9), subsequently. Rifr mutation influenced on virulence manifestation at albino mice and antigendeficient variants with Fra- and Fra-Tox- phenotype. In every group of strains highly virulent subcultures were registered. Resistance to nalidixic acid mainly was not associated with virulence loss. Nalr mutants of parent and antigenmodified mutants were cross resistant to fluoroqinolones (ciprofloxacin, ofloxacin, pefloxacin, lomefloxacin). LD50 for untreated albino mice did not differ from LD50, for mice treated with rifampicin (when mice were infected with strain resistant to rifampicin) or with nalidixic acid and fluoroquinolones (when animals were infected with Nalr mutants). Antigenmodified strains of plague bacteria and their Rifr, Nalr mutants were able to overcome specific immune reaction. The drugs should be used in synergic combinations (with aminoglycosides or cephalosporines of III generation) to prevent appearance of virulent strains resistant to rifampicin and fluroquinolones.     

Functional macrophage activity in infection with virulent and avirulent strains of the dysentery microbe

The effect of S. flexneri virulent and avirulent (vaccine) strains 2a on the cytoplasmic membrane of mouse macrophages has been studied by evaluating the action of these bacteria on the activity of 5-nucleotidase. The dynamics of the activity of 5-nucleotidase after the introduction of both virulent and avirulent strains has a phasic character with alternating rises and falls in the activity of this enzyme in comparison with the control. S. flexneri vaccine strain produces mainly a stimulating effect on the functional activity of peritoneal macrophages in mice, which is confirmed by a decrease in the activity of 5-nucleotidase; on the contrary, S. flexneri virulent strain- has mainly an inhibiting effect on the functional activity of peritoneal macrophages, which is confirmed by an increase in the activity of 5-nucleotidase in these cells. The comparative study of changes in the activity of 5-nucleotidase, following the introduction of S. flexneri, in mice, previously immunized with smallpox vaccine, and in intact mice has shown that the use of animals immunized with smallpox vaccine in the study of metabolic characteristics may lead to distortions in the results of the experiment.     

Thymidine phosphorylase, thymidine kinase and thymidylate synthetase activities in cerebral hemispheres of developing chick embryos

The changes in activities of thymidine phosphorylase (EC 2.4.2.4), thymidine kinase (EC 2.7.1.75) and thymidylate synthetase (methylenetetrahydrofolate:dUrd-5′-P C-methyltransferase, EC 2.1.1.-) in the cerebral hemispheres of developing chick embryos were determined and compared with the course of DNA synthesis and of natural cell death in this organ. Thymidine phosphorylase activity reaches a broad maximum at the 12th to 14th day of incubation, followed by a rapid decrease. Thymidine kinase and thymidylate synthetase activities are highest at the earliest time studied (day 10) and decrease until day 14, followed by an increase from day 14 to 16 and a further decrease from day 16 through 1 day post-hatching. The rate of DNA synthesis essentially follows these activities, but the increase at day 16 is not discernible. Our previous study revealed high DNA synthesis at day 10, with natural cell death concurring on days 12-14, followed by another peak after day 16 (glial proliferation) and a decrease after day 16. It appears that thymidine phosphorylase activity reaches a maximum (days 12-14) at the time of maximum cell death, which may be correlated with the degradative function of this enzyme. This was also the time for minimum activities of thymidine kinase and thymidylate synthetase; on the other hand, these activities reach a first (day 10) and second (day 16) maximum at the time of maximum DNA synthesis; this may be correlated with the synthetic functions of these enzymes.     

Resistance to levomycetin and activity of several enzymes in Escherichia coli and the agent of plague]

The authors compared the activity of acetyl-CoA-synthetase and of the enzymes belonging to the group of asparaginic acid in levomycetin sensitive and resistant strains of Y. pestis and E. coli. There were revealed marked differences in the activity of aspartase, fumarase, synthetase and desamidase of L-asparagin, and also of the enzyme activated by acetate in the E. coli strains with plasmide resistance. Transmission of R-factor to the pestis was accompanied by decomposition of L-asparadein, formation of AC-CoA. At the same time transformation of L-asparaginic acid catalyzed by aspartase remained on the same low level in the sensitive pestis cultures and their variants with the R-factor. When the resistance was controlled by chromosomal resistance markers, the activity of the enzymes providing formation of L-asparagic acid, its amide and L-malic acid showed no significant change. In chromosomal type of resistance in the mutants of pestis and E. coli the acetyl-CoA-synthetase reaction was as a rule somewhat increased.     

Ofloxacin efficacy in the prophylaxis and treatment of experimental plague due to antigen complete and defective strains of the pathogen]

Strains of the plague microbe, antigen complete and defective by fraction I and mouse toxin had the same in vitro susceptibility to ofloxacin (MIC 0.08 mg/L). The drug was superior in its activity to pefloxacin and especially nalidixic acid. In the experiments with albino mice (prophylaxis, 5 days) the ofloxacin efficacy was lower when the infection was due to the plague microbe strains deprived of the ability to produce fraction I and mouse toxin, evident from a statistically significant increase of the drug ED50 and a decrease of the animal survival percentage. When used in the doses corresponding to the human average daily doses, ofloxacin provided effective animal protection (80 to 100 per cent survival) after the prophylaxis for 7 days and the treatment of the plague infection irrespective of the strains, complete or antigen changed. However, when the infection is due to the antigen changed strain, ofloxacin should be used in the maximum daily doses at least for 7 days.     

Sensitivity to the differential dose of erythromycin and oleandomycin of various holarctic strains of tularemia microbe isolated in the Kazakh SSR]

The presence of strains of holarctic race of tuleremia microbe, both sensitive and resistant to the differential doses of erythromycine and oleandomycine was revealed at the territoryn of Kazakh SSR. The existence of such strains does not permit to differentiate them into geographical races by the sensitivity to erythromycine and oleandomycine.     

Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants

Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.     

Use of monoclonal antibodies to plague microbe antigens at the early stage of infection in white mice for enhancement of the late streptomycin and doxycycline treatment]

It was demonstrated that use for prophylaxy (after 5 h of infection) or for treatment (after 24 h after infection) of the monoclonal antibodies mixture to specific epitops of capsule antigen (fraction 1), lipopolysacharide, murine toxine can prevent development of plague pathogen at 100 of mice infected by approximately 1000 LD50 Yersinia pestis 231. 5-day course of prophylaxy by monoclonal antibodies provided survival of 50 per cent animals. Subsequent use of fraction 1 antigen for 5 days followed by treatment with streptomycin or doxycycline at 6-7-8-9-10 days after infection with Y. pestis 231 prevented infection manifestation at 80 per cent of animals, etiotropic therapy started at the same period was ineffective. When white mice were infected with Y. pestis 231 Fra-, with deleted ability to produce capsule antigen (fraction 1) 80% level of efficacy can be provided by subsequent administration of antibodies to fraction 1 combinated with lipopolysacharide, murine toxine and streptomycin. Use of monoclonal antibodies followed by doxycycline was ineffective.     

Expression, purification and antimicrobial activity of puroindoline A protein and its mutants

Wheat puroindoline proteins, PINA and PINB, play key roles in determining wheat grain hardness as well as in defending the plant against pathogens. PINA has much greater membrane-binding property and antimicrobial activity because it contains more tryptophan residues in the unique tryptophan-rich domain (TRD). In order to obtain proteins with higher antimicrobial activity, mutants of PINA containing two or three copies of TRD, designated ABBC and ABBBC, respectively, were constructed and expressed in E. coli Rosetta-gami (DE3). Metal affinity chromatography was used to purify the soluble affinity-tagged recombinant proteins. The secondary structures of the recombinant proteins were predicted by the online program Protein Homology/analog Y Recognition Engine v2.0 and experimentally assessed using circular dichroism. Minimum inhibition concentration tests and fluorescence microscope analyses were employed to evaluate the antimicrobial activities of the mutants. The results showed that the purified recombinant ABBC was correctly folded and presented significantly higher antimicrobial activities against E. coli and S. aureus than wild-type PINA, suggesting its potential use as an antimicrobial agent. The results also confirmed that TRD is a determinant of the antimicrobial activity of PINA and demonstrated that it is feasible to enhance the antimicrobial activity of PINA by adding one copy of TRD.     

Nucleotide pyrophosphatase activity in dry and germinated seeds of Triticum,and its relationship to general acid phosphatase activity

A cytochemical investigation has been made of nucleotide pyrophosphatase activity in dry and germinated seeds of Triticum, and its distribution compared to that of general acid phosphatase reactions seen with naphthol AS-BI phosphate and p-nitrophenylphosphate as substrates. Acid phosphatase activity was present in the cytoplasm and in channels through the walls of the aleurone cells in both dry and germinated seeds. The cytoplasmic activity was even more marked with nucleotide pyrophosphatase which was almost entirely absent from the cell walls. Nucleotide pyrophosphatase was active in all endosperm cells but particularly in some cells adjacent to the aleurone layer. In addition, all cells of the scutellum and embryo were positive for nucleotide pyrophosphatase activity, especially the developing fibres and xylem elements of leaves and coleoptiles, mature leaf xylem and phloem elements, scutellar provascular and vascular tissues and the epidermis of dark grown coleoptiles.Abbreviation GA₃ gibberellic acid     

Sensitivity of various races of the tularemia microbe to erythromycin, oleandomycin and polymyxin]

Sensitivity of 66 strains of the tularemia microbe to erythromycin, oleandomycin and polymyxin was studied. It was shown that all the strains were resistant to polymyxin. The strains of the non-artic and Central Asiatic races and 64 per cent of the strains of the holarctic race were resistant to erythromycin and oleandomycin.