[Binase penetration into alveolar epithelial cells does not induce cell death].

Microbial ribonucleases possess a broad spectra of biological activities, demonstrating stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. The mechanisms of their penetration into the cells are not clear so far. This research is aimed to the study of Bacillus intermedius RNase (binase) penetration in alveolar lung epithelial cells--pneumocytes of type II. Using immunofluorescence we have shown for the first time have internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in pneumocytes MLE-12, which also derived from type II cells. However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. The obtained results testified the higher sensitivity of tumor cells towards binase compared with normal cells, and also showed that penetration of the enzyme into alveolar cells did not directly correlated with the cell death.

[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.

[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.

[Competitive immunochemical determination of antigens using conjugates containing Co(II) and Ni(II)]. / Konkurentnoe immunokhimicheskoe opredelenie antigenov s pomoshch'iu kon"iugatov, soderzhashchikh iony Co(II) i Ni(II).

A new variant of competitive heterogeneous immunoassay for certain proteinaceous antigens has been developed. The assay is based on the use of the target protein conjugated with Co(II) or Ni(II) ions and immobilized Abs. The effect of catalytic hydrogen release allows quantitation of the metal ion labels by voltammetry at the final step of the assay. The conjugates have been characterized by spectrophotometry, voltammetry, atomic adsorption spectrometry, and nuclear magnetic relaxation. Based on the use of the conjugate RNase-diethylenetriaminepentaacetic acid-Co(II) (10:4:4), a competitive immunoassay for RNase has been developed, detecting the target protein in the range 2 x 10(-2)-2 x 10(-4) mg/ml.

[Noncompetitive immunochemical determination of ribonuclease using transition metal ions and the effect of catalytic hydrogen release]. / Nekonkurentnoe immunokhimicheskoe opredelenie ribonukleazy s ispol'zovaniem ionov perekhodnykh metallov i éffekta kataliticheskogo vydeleniia vodoroda.

A noncompetitive variant of immunochemical ribonuclease (RNase) determination has been developed, involving the use of Co(II) as a label. A variety of approaches to labeling the immunological reagent with the metal have been assessed. In the variant proposed, catalytic hydrogen release was used as a means of detecting the label, the amount of which was proportional to RNase concentration. Conditions making it possible to record catalytic hydrogen release fluxes were determined. In the presence of RNase, the electrocatalytic effect was maximum at a concentration of Co(II) in the ammoniac buffer, equal to 2 x 10(-4) M (pH 10.0). The dependence was linear in the range 4-2000 ng/ml RNase concentrations (threshold concentration, 2 ng/ml).

Factors influencing the cellular location of proteolytic enzymes of Bacillus intermedius.

Thiol-dependent serine proteinase and glutamylendopeptidase of Bacillus intermedius 3-19 being prevailing enzymes in the total pool of extracellular proteinases (95%) of this microorganism in catalytic active form were detected on the membrane of the cells. Production of these enzymes was maximum on the medium containing inorganic phosphate and gelatin and decreased 2-4-fold on the medium with glucose and lactate. The level of the activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused essential increase in extracellular glutamylendopeptidase activity and promoted the release of membrane-bound enzyme into cultural fluid. Proteolytic activity was detected in cytoplasm also. Proteinases localized in cytoplasm were shown to differ in properties from those secreted.

[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.

[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.

[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.

[Clinical regularities of the interaction of pancreatic and microbial RNase with cells in vitro]. / Kineticheskie zakonomernosti vzaimodeistviia pankreaticheskoi i mikrobnoi RNKaz s kletkoi in vitro.

The kinetics of interaction of exogenic RNAses with isolated cell culture of the human amnion FL was studied in a mathematical model. It was shown that by a number of kinetic parameters the pancreatic RNAse had higher affinity to the cells which quite agreed with higher cytotoxicity of the enzyme as compared to that of the microbial RNAse in regard to the cell culture.

[The effect of polymeric modification on the biological activity of pancreatic ribonuclease]. / Vliianie polimernoi modifikatsii na biologicheskuiu aktivnost' pankreaticheskoi ribonukleazy.

The effect of modification of dextran on pharmacokinetic properties of pancreatic RNAse and on its ability to suppress the proliferation of cells has been studied. It has been shown that the basic contribution to biological activity of polymer form of RNAse is making by azo-bonds which are forming in the process of chemical bonding of the protein with dextran support.

[Structural and antigenic differences of pancreatic RNAse preparations modified by dextran ethers in an azo-combination reaction]. / Strukturnye i antigennye razlichiia preparatov pankreaticheskoi RNKazy, modifitsirovannoi éfirami dekstrana metodom azosochetaniia.

Preparations of pancreatic RNAase modified by dextrane derivatives were obtained in an azocombination reaction. The UV absorption spectra and amino acid analysis of the preparations revealed quantitative and qualitative differences in the sites of the enzyme binding to the polymeric matrix depending on modification conditions. The observed differences in the binding sites of modified RNAase may be related both to the differences in the primary structure or even in the secondary and ternary structure of the enzyme. The latter observation was confirmed in studies of the antigenic properties of modified preparations. The interrelationship of these preparations with antibodies raised against native RNAase and the comparison of the degree of their antigenicity suggest the influence of modification on the antigenic properties of the protein. The number of binding sites of the enzyme to the support can be determined, which would neutralize the antigen-stimulated effect of the support due to the screening of protein antigenic determinants.

[Modification by azocombination of the catalytic properties of ribonucleases]. / Vliianie modifikatsii azosochetaniem na kataliticheskie svoistva RNKaz.

Using pancreatic RNAase and RNAase from Act. rimosus as models, the effect of modification by azocombination on the catalytic properties of enzymes were studied. It was shown that RNAases binding to soluble dextran did not cause any significant changes in their major catalytic properties, when polymeric RNA was used as a substrate. At the same time, the physico-chemical properties of the modified enzymes may result in changes in the catalytic properties in a reaction with low molecular weight substrates. Evidence for this observation can be obtained from the increase in the synthetic activity of modified pancreatic RNAase as compared to the hydrolase activity in the dinucleotide synthesis reaction.

[Modification of nucleases by soluble derivatives of polysaccharides]. / Modifikatsiia nukleaz rastvorimymi proizvodnymi polisakharidov.

Preparations of pancreatic RNAase, ribonuclease Act. rimosus and nuclease Ser. marcescens covalently bound to water-soluble derivatives of polysaccharides (m-aminobenzyl-oxymethyl ether of dextran and mannan, 4beta-oxyethylsulphonylanisol, 4beta-oxyethylsulphonylaniline, 3-Cl-2-oxypropyl ethers of dextran and dialdehydedextran) have been obtained. The yields and thermal stability of immobilized nucleases depend both upon the amount and nature of the functional groups which activate the polysaccharide. Polysaccharide aminoaryl ethers capable of binding to proteins by azocoupling present special interest in view of their utilization as modifying baskings. Regulation of effectiveness of the azocoupling reaction by means of structural changes of the diazocomponent and the reaction conditions were shown. All this allows to obtain immobilized enzymes with different physico-chemical properties.