[EFFECT OF EXTRACHROMOSOMAL ELEMENTS OF HEREDITY ON TOXIC PROPERTIES OF YERSINIA PESTIS].

AIM: Elucidation of the role of extrachromosomal elements of heredity in manifestations of toxic properties of Yersinia pestis. MATERIALS AND METHODS: The study was carried out in vac- cine strain Y pestis EV76 (pMT1, pCD1, pPCP1) and non-plasmid variants of vaccine EV76 (pMT1⁻, pCD1⁻, pPCP1⁻) and virulent 231 (pMT1⁻, pCD1⁻, pPCP1⁻) strains of Y pestis. Presence of functionally active form of lipopolysaccharide (LPS) in.the incubation medium of the bacteria was evaluated via toxicity of supernatant of Y pestis for intactanimals (infec- tion-toxic shock) and mice sensitized by D-GalN. RESULTS: 37°C cultures of Y pestis EV76 containing a full amount of plasmids were established to release LPS into the environment. Non-plasmid variants of both vaccine and virulent strains of Y.pestis pMT-, pCD1-, pPCP1- do not have this ability. Separation of LPS from cell wall was detected in live bacteria of plague infectious agent. This process is assumed to be coupled with translocation of proteins coded by pMT1, pCD 1, pPCP 1 plasmids from the cell into the environment. CONCLUSION: Functional inter-connection between extrachromosomal elements of heredity and toxic activity of Y pestis LPS is established for the first time.

[Anti-nitrosative system as a factor of malignant tumor resistance to cytotoxic effect of nitrogen monooxide].

The inhibitory action of binuclear dinitrosyl iron complexes with glutathione on the growth of implanted solid tumor in BDF1 mice bearing Lewis lung carcinoma cells was found. The effect was induced by intraperitoneal injection of the binuclear dinitrosyl iron complexes to mice at a dose of 200 µM/kg daily on days 1-5 and 7-11. At the binuclear dinitrosyl iron complexes: free glutathione ratios of 1:1; and 1:10 in solutions, the inhibitory effect of the DNICs reached the level of 70% and 85%, respectively. When B-DNICs were not further infused, intensive tumor growth, a more rapid rate of tumor growth than control, was observed. The selective accumulation of DNICs as well as iron nitrosyl complexes of heme-containing proteins in tumors were detected by EPR method. The latter were found also in the tumors in control animals. Tumor growth delay in course of B-DNIC administration to the mice is supposed to be due to the elaboration of anti-nitrosative defense in tumor tissue in response to the action of NO released from B-DNIC.

[Anti-Tumour Activity of Dinitrosyl Iron Complex with Glutathione and S-Nitrosoglutathione Preparations: Comparative Studies].

The anti-tumor activity of the binuclear form of dinitrosyl iron complexes with glutathione against Lewis lung carcinoma, found earlier upon intraperitoneal administration of the complexes, was also observed when this preparation was injected subcutaneously. A 100 µM/kg subcutaneous dose of the complex being used daily (as calculated per one iron atom in binuclear dinitrosyl iron complexes) for 10 or 15 days, inhibited the tumor growth by 43%. The effect was observed during the first two weeks after tumor transplantation. After that, the tumors began to grow at the rate equal to or even higher than that one for control animals. The mean survival time for treated mice exceeded the control values by 30%. Binuclear dinitrosyl iron complexes administered intraperitoneally was also effective against Ca-755 adenocarcinoma. However, in this case the mean survival time for treated animals increased only by 7%. The anti-tumor activity of S-nitrosoglutathione against Lewis lung carcinoma growth inhibition by 70% and Ca-755 adenocarcinoma growth inhibition by 90% was also shown. However, unlike binuclear dinitrosyl iron complexes the anti-tumor effect of S-nitrosoglutathione decreased when a daily dose of the compound increased (from 200 to 400 µM/kg) The initial anti-tumor effect of binuclear dinitrosyl iron complexes and S-nitrosoglutathione is suggested to be due to NO released from both compounds. A subsequent suppression of the effect is determined by the development of anti-nitrosative and anti-oxidant defense systems in tumors.

[Anti-tumour activity of dinitrosyl iron complexes with glutathione].

The anti-tumour dose-dependent effect of binuclear dinitrosyl iron complexes with glutathione as NO donors on solid tumour in the mouse Lewis lung carcinome was detected. The complexes being injected at the doses of 21, 42, 105 mg/kg daily during 10 days blocked completely the development of the tumour for the first week after tumour cell implantation into animals. After that, the part of tumour cells which remained in intact alive state began to grow at the rate equal to that for control animals. The effect was proposed to be caused via the formation of the anti-nitrosative defense system in the cells as a response to NO attack to cells. It was also hypothesized that this system can be inactivated by higher doses of dinitrosyl iron complexes. The data were obtained which were in line with the hypothesis.

[Polyacrylates of noble metals as potential antitumor drugs].

The antitumor activity of polyacrylates of the noble metals containing argentum (argacryl), aurum (auracryl) and platinum (platacryl) has been studied using experimental murine solid tumor models (Lewis lung carcinoma and Acatol adenocarcinoma). It has been found that polyacrylates of the noble metals are capable of inhibiting tumor development by 50-90% compared to control. Auracryl that inhibites the growth of Lewis lung carcinoma and Acatol adenocarcinoma by 80 and 90%, respectively, compared to control is the most efficient among the tested compounds and can be recommended for the further profound preclinical studies.

[The autoagglutination factor of plague pathogen--cross-reacting antigen].

An antigen similar to the autoagglutination factor (AF) of plague pathogen in immunochemical specificity was sought in 22 bacterial species. For this, an immunoglobulin anti-AF diagnosticum that is the sheep erythrocytes sensitized with rabbit immune serum to the AF preparation isolated from the plasmid-free variant of the Yersinia pestis strain EV76. The bacteriological study applying a passive hemagglutination assay revealed AF-like antigens not only in all study strains (n = 30) of Y. pestis, but also in Yersinia pseudotuberculosis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus vulgaris. These bacteria were used to prepare AF = like antigen preparations that reacted with rabbit anti-AF serum in dot blot immunoassay. Also, as Y. pestis, AF-like antigens in polyacrylamide gel were detected as multiple protein bands that differed in mobility in Yersiniae and heterologous bacteria. Differences were found in the properties of AF-line antigens of 5 species of bacteria (sensitivity to temperature and formalin, binding to the cell surface, which enabled differentiation of serological reactions caused by AP-like antigens of other bacteria. Thus, Y. pestis AF is a cross-reacting antigen that, despite its immunochemical similarity with AF-like antigens of other bacteria, was ascertained to differ from them in properties. The findings are of interest for searching for new diagnostic tests to detect the Cafl- strains of the plague pathogen and for differentiating the causative organisms of plague and pseudotuberculosis.

[Immunochemical characteristics of the autoagglutination factor of plague pathogen].

Immunochemical studies were performed to characterize the autoagglutination factor (AF) isolated from the plasmid-free variant of Yersinia pestis EV76 strains. The preparation based on the capsule Cafl antigen isolated from the triplasmid variant of the same strain was used for comparison. AF and Cafl are the surface protein antigens of the plague pathogen, which are similar in their capacity for self-aggregation and in the molecular mass of a subunit (about 17 kDa). However, unlike Cafl, FA gives rise to protein aggregates resistant to 2-mercaptoethanol and sodium dodecyl sulfate. FA and Cafl were shown to be immunochemically different antigens, the antibodies to which were present in the plague hyperimmune equine serum. Thus, the new surface antigen of the plague causative organism, which induces the generation of antibodies during immunization with live bacteria of the vaccine Y. pestis strain, has been discovered.

Isolation and characterization of autoagglutination factor of Yersinia pestis Hms- cells.

An approach for isolation of an autoagglutination factor (AF) from Hms(-) cells of the plague agent has been developed. Purified AF has been obtained and characterized in physicochemical properties. The AF is found to be a complex of a 17.5-kD protein with a low molecular weight peptide component, which binds iron ions and shows siderophore activity. This low molecular weight component is responsible for hydrophobic properties and immunochemical activity of the AF, as well as for its ability to interact with the plague diagnosticum L-413c bacteriophage.

[Identification of the autoagglutination factor of Hms- cells of the plague agent].

A search for cellular components responsible for autoagglutination (AA) in broth and salt solutions of Hms- cells of the plague agent Yersinia pestis was performed. The AA- mutants were obtained using vaccine strain Y. pestis EV76 derivative containing one species-specific plasmid pYP. The mutants were shown to differ from the parent strain by the decreased surface hydrophobicity, insensitivity to plague diagnostic L-413c bacteriophage and negative haemagglutination reaction with antibodies to F1 capsular substance of the plague agent. The mutants did not differ from the parent strain by electrophoretic mobility and immunochemical activity of LPS but were characterized by the absence of a 17 kDa protein on the cell surface. The AA+ cells that lost this protein after weak alkali extraction were less hydrophobic and failed to express AA in 0.5 M ammonium sulfate. After the extraction, the cells lost the ability to neutralize L-413c and to react with the anti-F1 antibodies, while both activities as well as 17 kDa protein were detected in the extracts. Thus, the 17 kDa protein is suggested to be a hydrophobic surface antigen which acts as a receptor of the L-413c bacteriophage and represents an AA factor of Hms- cells of Y. pestis.

Inhibition of tumor growth with ultralow doses of doxorubicin under experimental conditions.

Antitumor activity of ultralow doses of cytostatic doxorubicin was studied on BDF1 mice with Lewis lung carcinoma. The preparation was injected intraperitoneally in single doses of 10(-5), 10(-10), 10(-15), and 10(-20) M on the next day after tumor inoculation. The effect of ultralow doses was compared with that of a standard therapeutic dose of doxorubicin (8 mg/kg, 1.4 x 10(-3) M). Doxorubicin in ultralow doses produced an antitumor effect comparable with that induced by the preparation in standard doses. On day 12 after administration of doxorubicin in ultralow and standard doses, tumor size in mice did not exceed 20% of the control level.

[The sensitivity of experimental tumor systems to ultra low doses of doxorubicin]. / Chuvstvitel'nost' éksperimental'nykh opukholevykh modelei k sverkhmalym dozam doksorubitsina.

Biological effect of doxorubycin in standard (10(-3) mol/l) and ultra low doses (10(-5)-10(-20) mol/l) against some "signal" animal tumor systems--Lewis lung carcinoma, 755 adenocarcinoma, B-16 melanoma, Ehrlich carcinoma and L1210 leukemia was studied. The all models were very sensitive to the action of the drug in standard dose. Solid tumors' growth inhibition by 80-95% as well as increasing in life span of mice with L1210 leukemia by 86% in comparison with control and surviving of animals with Ehrlich carcinoma had been revealed. It had been shown that the drug in the area of ultra low doses occurred the following effects: inhibition of Lewis lung carcinoma growth by 80-95% compared to control after administration of the all tested ultra low doses; increasing of the life span of the animals with Ehrlich carcinoma and L1210 leukemia by 86-123% and 6-23%, correspondingly, upon the action of all tested ultra low doses; inhibition of B-16 melanoma growth by 50 and 70% after administration of the drug in doses 10(-20) mol/l and 10(-5) mol/l, correspondingly as well as deceleration of 755 carcinoma growth by 40% compared to control after action of the drug in the dose 10(-20) mol/l; stimulation of the B-16 melanoma growth by 20% relative to control after 10(-10) mol/l dose injection and enhancement of tumors sizes by 20-60% above control levels as a result of treatment of mice with 755 carcinoma by the drug in such ultra low doses as 10(-5) and 10(-15) mol/l. So, it was found that all tested tumor systems revealed certain sensitivity to the some ultra low doses of the drug. At the same time it was shown that doxorubycin in ultra low doses displayed alternative character of its biological effect, directivity of which varied according with the dose level and tumor strain.

[Antitumor effect of joint action of low intensity electromagnetic fields and ultra low doses of doxorubicin]. / Protivoopukholevaia éffektivnost' sovmestnogo vozdeistviia nizkointensivnogo élektromagnitnogo polia i sverkhmalykh doz doksorubitsina.

Combined action of a low intensive physical factor and a chemotherapeutic agent in ultralow doses against Lewis lung carcinoma was studied. Antitumor activity of low intensiwe electromagnetic field was expressed as inhibition of tumor growth at 60% compare to control. Ultra low doses of doxorubicin as well as its standard dose resulted in inhibition of tumor growth by 60-70% in comparison with control. Joint action of both factors leaded to increasing in the antitumor effect that reached such level of tumor growth inhibition as 85% relative to control.

[Study of PGM mutation mechanism in Yersinia pestis (plague pathogen) vaccine strain EV76]. / Izuchenie mekhanizma vozniknoveniia PGM--mutatsii u vaktsinnogo shtamma vozbuditelia chumy Yersinia pestis EV76.

Yersinia pestis vaccine strain EV76 is a mutant of the virulent strain which has lost the pigmentation phenotype (Pgm+). This phenotype includes three characteristics: it absorbs pigments from agar media (Hms+), produces a siderophore yersiniabactin (Ybt+), and causes a lethal disease after subcutaneous inoculation of laboratory animals (Vir+). These characteristics are lost simultaneously after high frequency spontaneous deletion of 10 kB fragment of chromosomal DNA, termed the pgm locus. We compared the pgm locus-associated genetic and phenotypical properties of the vaccine strain with those of a typical Pgm- deletion mutant of a virulent strain. The results indicate that Pgm- phenotype of the vaccine strain results not from the deletion of the pgm locus, but from the insertion inactivation of the genes located in this locus. In contrast to the deletion mutant, the vaccine strain carries sequences detected by hybridization and PCR, which are complementary to the pgm locus genes. Moreover, the vaccine strain differed from the deletion mutant by a low level of Hms+ expression, a slower rate of cell death under iron-chelated conditions at 37 degrees C, "residual virulence" upon subcutaneous inoculation, and capacity to form revertants which restore the characteristics of Pgm+ phenotype after cell growth at 12 degrees C.

[Taxotere and methylnitrosourea: experimental appraisal of combination chemotherapy]. / Taksoter i nitrozo metilmochevina: élsperimental'naia otsenka éffektivnosti sovmestnogo primeneniia.

The effectiveness of taxotere and methylnitrosourea (MNU) combined application against murine P388 leukemia has been studied. Antitumor drugs were administered separately or in combination in doses of 50, 60 or 70 mg/kg per day, i/p, beginning from day 1 after tumor transplantation. Combined administration was shown to potentiate antitumor effect, which also largely depended on schedule. Survival increase varied within 50-130% of control values. Optimal effect was observed with the following schedules: taxotere + MNU, 24-hour interval between injections, and MNU-taxotere, 4-day interval between injections. Synergism was in evidence since the therapeutic effect of combination treatment was significantly higher than that of either drug administered alone.

[Sensitivity of human melanoma xenografts to nitrosoalkylurea antineoplastic agents]. / Chuvstvitel'nost' ksenograftov melanomy cheloveka k protivoopukholevym preparatam klassa nitrozoalkilmochevin.

We studied the sensitivity of human melanoma (Bro strain) xenografts to drugs of the nitrosoalkylurea (NAU) class: nitrosomethylurea (NMM), karmustin (BCNU), nimustin (ACNU), nitrulin, and ADEKO. High antitumor activity of NAM was shown when the drugs were applied not only at the early, but also at the late stages of tumor progression (tumor mass 400 and 1200 mg, respectively). The therapeutic effect of the drugs was estimated with the use of criteria characterizing the kinetics of tumor regression, increased life span, and survival of treated animals. After early administration of the drugs (Day 4 after tumor transplantation), 67% and 50% of animals survive under the influence of nitrulin and ACNU, respectively, while the rate of tumor regression increased in the sequence nitrulin < karmustin < NMM < ACNU. After late administration (11 days after tumor transplantation), NMM was most effective at increasing survival (35% of survived animals by 35 days of observation), while the rate of tumor regression increased in the sequence ADEKO < NMM < karmustin < nitrulin < ACNU.

[Kinetics of development of human melanoma xenografts in immunosuppressed animals]. / Kinetika razvitiia ksenograftov melanom cheloveka na immunosupressirovannykh zhivotnykh.

An experimental model of development of the human tumor xenografts (melanomas HCMB and Bro) in immunosuppressed phenotypically normal animals (CBA/Ca mice) was developed. Optimal conditions were established for immunodeficiency induction in the animals. Kinetics of development of the xenografts in the immunosuppressed animals was studied. The level of 0(6)-alkyl-guanine-DNA-alkyltransferase, an enzyme responsible for repair of DNA damage in the tumor cells induced by chemotherapeutic agents (nitroalkylurea), was determined.

[The sensitivity of tumor cells to low doses of antitumor preparations]. / Chuvstvitel'nost' opukholevykh kletok k nizkim dozam protivoopukholevykh preparatov.

The anticancer activity of water solutions of nitrosomethylurea, adriblastin and cisplatin at low and extra low concentrations (to 10(-15) M) was studied in the mice with experimentally induced tumor. Single and multiple administration of these drugs at extra low doses changed the life duration. Nitrosomethylurea (10(-15) M) increased the life duration by 20-86%. Nitrosomethylurea at extra low concentrations modifies the organism and tumor sensitivity to subsequent action of a high dose of this drug.

[The use of mini-Mu-replicons for cloning of various structural genes of Yersinia pestis]. / Ispol'zovanie mino-Mu-replikonov dlia klonirovaniia nekotorykh strukturnykh genov chumnogo mikroba.

The possibility to clone the structural leu gene of Yersinia pestis in vivo using the mini-Mu bacteriophages with the inserted plasmid replicones has been demonstrated. The E. coli K12 transductants having obtained the Leu(+)-marker within the cloned 4.8-21 kb fragments stably inherited the leu-gene within the autonomous mini-Mu replicones. The possibility to clone other Yersinia pestis genes by the same technique has been demonstrated.

[14Co-dimetinur distribution in mice in relation to the stage of the development of tumor process]. / Raspredelenie 14Co-dimetinura v organizme myshei v zavisimosti ot stadii razvitiia opukholevogo protsessa.

Pharmacokinetics of the antitumour agent 14CO-dimetinur (100 mg/kg) after oral administration to the intact mice and those with solid leukemia P 388 is characterized by its rapid delivery to organs and tumours with the achievement of maximum radioactivity 5 hours later and the further gradually decline during 4 days. The increased accumulation of the 14CO-products in kidneys and their retarded output from the brain and lungs against a background of the relatively equal distribution of radioactivity between other tested organs have been established. The same level of carbamoylated products in large tumours (the 16th day after leukemia transplantation) as well as in small tumours (the 9th day after inoculation) is in agreement with the conservation of the initial marked inhibitory effect of the drug against advanced tumours.

[The pharmacokinetics of the antitumor preparation of dimetinur]. / Farmakokinetika protivoopukholevogo preparata dimetinur.

The pharmacological disposition of 1,3-dimethyl-1-nitrosourea (dimetinur) in intact rats and animals with Walker carcinosarcoma, glioma 2211, colon adenocarcinoma was studied by the colorimetric assay using an oral drug dose of 100 mg/kg. Computer analysis of data was based on a single-compartment model using the area under the concentration-time curve (S) and the intact drug half-life (t1/2) as main pharmacokinetic parameters. The highest level of the drug (S) was observed in tumour and brain tissues on an equality with drug distribution between blood, spleen, kidney and lungs. The half-life of the dimetinur removal from blood exceeds the known values for certain active NAM type. The antitumour activity of the drug against the studied tumours correlates positively with pharmacokinetic parameters for the tumours (S and 1/tmax).