[Induction of the unculturable state in Escherichia coli K12 with 2,4,6-trinitrotoluene].

Abstract-The toxic effect of high (200 mg/l) 2,4,6-trinitrotoluene (TNT) concentrations on Escherichia coli K12 cells in the absence of exogenous nutrient sources (incubation in 0.5% NaCl) was shown to manifest itself in the transfer of the culture to an unculturable but viable state; its reversal depends on the duration of culture contact with the xenobiotic and the conditions of cell recultivation. The likelihood that cell succession to death forms the basis of the physiologo-biochemical mechanism of the unculturable state in Escherichia coli K12 population under conditions of combined toxic and starvation stress is discussed.

[Inhibition of functional activity of macrophages in vitro by dimer RNase Bacillus intermedius].

The influence of cross-linked by dimethylsuberimidate dimeric RNAse from Bacillus intermedius on peritoneal rat macrophages has been investigated in vitro. It has been shown that dimeric RNase with concentrations of 0.5-40.0 mg/ml decreases the functional activities of macrophages. This is manifested in the inhibition of the phagocyte function of macrophages and suppression of the fusion of phagosomes with lysosomes. The change in the cytoplasmatic membrane surface structure induced by the dimers, which is stronger than that induced by monomers, has been demonstrated using atomic force microscopy. The role of membrane properties modification in the inhibition effect of RNase dimers on the functional activities of macrophages is discussed.

[Electrostatic interaction of pancreatic and microbial ribonucleases with macrophages in vitro].

The interaction of RNase A and RNase Bacillus intermedius with peritoneal macrophage of rats has been studied. To estimate the efficiency of this interaction the spontaneous chemiluminescence and induced by phagocytosis chemiluminescence of macrophages were investigated. It has been shown that electrostatic interaction of enzyme proteins with negatively charged cell membrane makes substantional contribution to the development of chemiluminescence reply of macrophages. The RNase A, which is more basical than the other (i. e. it has larger value of pI) is less effective with respect to macrophages. The calculation of total charge and dipole moment of pancreatic and microbial RNases showed that the efficiency of interaction between protein polycation and a cell was not connected with pI and depended on the charge and its distribution on the surface of protein molecule at the given pH value.

[Assessing the toxic effect of 2,4,6-trinitrotoluene on cells of escherichia coli K12 by flow cytofluorometry].

The magnitude of transmembrane potential delta psi in cells of Escherichia coli K12 was determined by the method of flow cytofluorometry for different phases of growth. It was large in the log phase, whereas in the lag and stationary phases, the population was shown to consist of two subpopulations with low and large values of delta psi in cells. In the presence of 2,4,6-trinitrotoluene (TNT), this bimodal distribution of delta psi over the population was observed during the entire growth period until TNT was almost completely eliminated from the cultivation medium (to a concentration of 18-20 mg/l). The mean value of delta psi in cells of the population grown in the presence of TNT was substantially smaller than that in controls due to the larger size of the subpopulation with a low value of delta psi. Upon elimination of TNT, the distribution of delta psi in cells of the culture became unimodal and close to that in the control culture in the early log phase of growth. These findings are discussed from the standpoint that considers heterogeneity of the culture of Escherichia coli K12 as a mechanism of its adaptation to the presence of xenobiotics.

[Features of the toxic action of 2,4,6-trinitrotoluene on Escherichia coli K12].

2,4,6-Trinitrotoluene present in a culture of Escherichia coli K12, at a concentration of 200 mg/l, caused a decrease in the total cell population and the population of colony-forming units, increased permeability of the external lipoprotein envelope, and increased the refractive index of cells. The shape of some cells changed from rod-like to coccoid, and cell size decreased. The specific rate of glucose consumption and the content of NADH (NADPH) in cells decreased. The changes of these morphological and physiological features were reversible, tending to normalize after reduction of 2,4,6-trinitrotoluene concentration in the course of cultivation.

[The influence of native ribonucleases and their modified derivatives on the functional activity of rat peritoneal macrophages].

The influence of native, hydrophobized and dimeric forms of ribonucleases (RNAse A and RNAse Bacillus intermedius) on the process of phagocytosis and fusion between lysosomes and phagosomes in rat macrophages has been studied. The effect of native RNAses depends on their concentration: comparatively low concentrations (0.5-50 microg ml(-1)) stimulate the phagocytosis and phagosome-lysosome fusion whereas high concentrations (above 75 microg ml(-1)) inhibit these processes. RNAses modified by surfactant oxanol-KD-6 and dimeric forms of RNAses possess only the inhibitory effect, which appears at concentration considerably lower than that of native enzymes. The stimulatory effect of native RNAses and the inhibitory effect of hydrophobized derivatives do not depend on the catalytic activity.

[Sensitivity of various escherichia coli strains to 2,4,6-trinitrotoluene].

The sensitivity of Escherichia coli strains K-12 and 055 to 2,4,6-trinitrotoluene (TNT) was found to correlate with the structural and functional properties of the outer lipoprotein membrane. The protective ability of the membrane of strain 055 is much lower than that of K-12. This is the cause of the greater sensitivity of 055 to the toxic action of TNT. High TNT concentrations (100-200 mg/l) suppressed the growth of 055, whereas K-12 grew at all TNT concentrations studied. Both strains adapted to high TNT concentrations by converting it by either nitroreduction or denitritation depending on concentration. The denitritation system of strain 055 started TNT degradation earlier than that of K-12.

[Effect of toxic concentrations of 2,4,6-trinirtotoluene on the physical properties and morphology of bacillus subtilis SK1]. / Vliianie toksicheskikh kontsentratsii 2,4,6-trinitrotoluola na fizicheskie svoistva i morfologiiu kletok Bacillus subtilis SK1.

It has been demonstrated for the first time that the toxic effect of 2,4,6-trinitrotoluene on the gram-positive strain Bacillus subtilis SK1 is accompanied by a decrease in the cell size and an increase in the refraction index (i.e., density) and thermostability of the culture. These data suggest that determination of kinetic parameters of bacteria growing under the conditions of toxic stress from optical density measurements may result in erroneous conclusions.

[Specific toxic effects of 2,4,6-trinitrotoluene on Bacillus subtilis SK1]. / Osobennosti toksicheskogo éffekta 2,4,6-trinitrotoluola v otnoshenii Bacillus subtilis SK1.

Using Bacillus subtilis SK1 as an example, it was demonstrated for the first time that 2,4,6-trinitrotoluene (TNT) transformation pathways change with TNT concentration. The growth of cultured B. subtilis SK1, delayed at 20 mg/l TNT (minimum toxic concentration), was resumed following TNT transformation. Aromatic amines were predominant metabolites detected in the culture medium at early stages of TNT transformation. The culture growth was completely inhibited by 200 mg/l TNT. As this took place, nitrites accumulated in the culture medium.

Effect of ribonuclease from Bacillus intermedius on human blood lymphocytes.

By using a rosette formation test the effect of ribonuclease Bacillus intermedius (RNase Bi) on T- and B-lymphocytes in human peripheral blood has been studied in vitro. The RNase effect on T-lymphocytes depends on its concentration: low concentrations (10(-6)-10(-2) microg ml(-1)) stimulate E-rosette formation whereas high concentrations (10 microg ml(-1)) suppress it. The amount of B-lymphocytes decreases under RNase Bi influence in all concentrations tested. RNase Bi like thymus hormones influence immature lymphocytes (0-cells) by inducing the surface expression of E-receptors what leads to rosette formation and, thus, contributes to lymphocyte differentiation. The increase in the amount of active T-cells which represent the mature cell population also confirms the participation of RNase Bi in T-rank lymphocytes differentiation processes. The RNase Bi effect on the human lymphocytes depends on its catalytic activity.

[Cytotoxic properties of pancreatic ribonuclease, modified by the surface active substance oxanol KD-6]. / Tsitotoksicheskie svoistva pankreaticheskoi ribonukleazy, modifitsirovannoi poverkhnostnoi aktivnym veshchestvom oksanolom KD-6.

Pancreatic RNase modified by the surface active substance oxanole KD-6 (OxRNase) was studied in respect to its cytotoxic action on cells. The studies included in vitro and in vivo tests with intravital staining of the cells by neutral red and the 3H uridine label, as well as the test with the preparation action on fusion of lysosomes and phagosomes. It was shown that in all the tests the hydrophobised RNase had a higher cytotoxic action versus the native enzyme. The analysis of the experimental data suggested that the cytotoxicity of the hydrophobised RNase was due to its action on the cell membrane structures including the lysosome membranes.

[Cytotoxic properties of the complex of the antineoplastic antibiotic bleomycetin and Bacillus intermedius ribonuclease]. / Tsitotoksicheskie svoistva kompleksa protivoopukholevogo antibiotika bleomitsetina i ribonukleazy Bacillus intermedius.

It was shown possible to change the cytotoxic properties of the antitumor antibiotic bleomycetin by its binding to Bacillus intermedius RNAse. The complexing lowered the antibiotic effect on DNA in the cells of the human amnion. At the same time the experiments with human red blood cells indicated that RNAse of B. intermedius in complex with bleomycetin-Fe(II) increased the antibiotic capacity for the cell membrane break down.

[Modification of Bacillus intermedius ribonuclease by dialdehyde dextran]. / Modifikatsiia ribonukleazy Bacillus intermedius dial'degiddekstranom.

Conditions for modification of Bacillus intermedius ribonuclease with dialdehyde dextran was optimized by Box and Wilson's method to achieve the enzyme with higher activity. The generalized parameter including the yield of enzyme protein and enzyme stability during modification was used. The yield of activity and preparation characteristics depended on a number of enzyme-carrier bonds.

[Chemical modification of RNAse from Bacillus intermedius. Effect on the cytotoxic properties of the enzyme]. / Khimicheskaia modifikatsiia RNKazy Bacillus intermedius. Vliianie na tsitotoksicheskie svoistva fermenta.

Modified derivatives of RNAse from Bacillus intermedius differing in the aggregate enzymatic, physico-chemical and physical properties were prepared. The cytotoxic properties of the compounds were tested by the intravital staining of FL cells with neutral red. It was shown that the native enzyme cytotoxicity correlated with the catalytic activity. However, it was found possible to lower the cytotoxicity of the catalytically active compounds by changing the enzyme aggregate properties.

[Changes in the size and morphology of cells under the influence of bacterial ribonuclease]. / Izmenenie razmerov i morfologii kletok pod vliianiem bakterial'noi ribonukleazy.

It has been found that a single interperitoneal injection of mice with NKLy ascite lympholeukosis cells and Bacillus intermedius RNAse or its derivative, obtained in result of activation of the active centre, on histidine induces an increase in size of cells cultured in vivo, the number of morphological changes in the cell cytoplasm, cell nucleus and, to a large extend, in the cell surface. A share of 2-3 nucleated cells increases as a result of both an incomplete mitosis and cell fusion. These changes do not depend on catalytic activity of RNAse. 24 hours following RNAse injection, both dimensions and morphology of the majority of cells returned to the norm. The trigger mechanism resulting in all the above mentioned changes is thought to be RNAse bringing about nonspecific irritation (reversible damage) of cells.

[Preparation and antiviral properties of Bacillus intermedius ribonuclease modified by chloranhydride of adamantanecarboxylic acid]. / Poluchenie i protivovirusnye svoistva ribonukleazy "Bacillus intermedius", modifitsirovannoi khlorangidridom adamantankarbonovoi kisloty.

Bacillus intermedius ribonuclease modified by the residue of adamantane carboxylic acid was prepared. When the cells of chick embryo fibroblasts infected by the fowl plague virus were exposed to the modified ribonuclease, the antiviral activity proved to be higher by comparison to that of the native enzyme. The chemotherapeutic index of the RNAse after the modification increased 4 times.

[Ribonuclease stimulation of nonspecific factors of protection in experimental animals]. / Stimuliatsiia ribonukleazami nespetsificheskikh faktorov zashchity v organizme éksperimental'nykh zhivotnykh.

It was shown that a single intraperitoneal administration of Bacillus intermedius RNAse to rats stimulated the activity of lysozyme and blood serum complement. A single intraperitoneal administration of pancreatic RNAse, Bacillus intermedius RNAse and its derivative selectively inactivated by the histidine active centre stimulated the metabolic activation of neutrophils as was shown by their ability to reduce tetrazolium nitroblue to diphormazone. The efficiency of the neutrophil stimulation by the RNAses was comparable with that of the microbial vaccine and did not depend on the catalytic activity of the RNAses.

[The mechanism of the antitumor action of Bacillus intermedius ribonuclease]. / Mekhanizm protivoopukholevogo deistviia ribonukleazy Bacillus intermedius.

In a concentration providing the maximum in vivo antitumor action, "Bacillus intermedius" RNAase had no in vitro cytotoxic effect on the cells of lympholeukosis NK/Ly. The enzyme did not inhibit the involvement of 3H-thymidine in the DNA biosynthesis in the tumor cells cultivated in vitro. A preliminary in vitro exposure of the cells to the enzyme did not influence their inoculation capacity. It was concluded that the mechanism of the enzyme antitumor action was not associated with the inhibition of the tumor cell viability. It was shown to be due to the activation of the intermediate effector system. The suggested mechanism of the mediated RNAse action on the tumor growth includes stimulation of the host immunity or removal/restriction of the tumor systemic action on the host.

[The modification of Bacillus intermedius ribonuclease by urea derivatives]. / Modifikatsiia ribonukleazy Bacillus intermedius proizvodnymi mochevin.

The results of the "Bacillus intermedius" RNAase modification by urea derivatives are presented. The modifiers synthesized for that purpose were the following: N-(4-chlorbenzoyl)-N'-benzolsulfonyl urea (M1) and N-(4-chlorbenzoyl)carbomoyl-epsilon-aminocapronic acid (M2). It was shown that RNAase modified by M2 stimulated the cell general metabolism by the test of the cell absorption of neutral red and had a more marked ability to disrupt RNA of the cell plasmatic membranes in comparison to that of the native enzyme.