Cytomorphologic assessment of the effect of Bacillus intermedius RNase on the organs of experimental animals]

Cytomorphological changes in the organs of laboratory animals after their treatment with RNAse from Bacillus intermedius were investigated. It was shown that the effect of RNAse on the thymus and spleen was evident from stimulation of the elements of the lympho and hemopoiesis while its effect on the liver manifested itself in increased stromal response of the liver and higher functional activity of the organ. The observed effects did not depend on catalytic activity of the enzyme.     

The isolation of highly purified bacillary ribonucleases from recombinant strains of Escherichia coli]

Active and inactive highly purified extracellular alkaline ribonucleases (barnase) from recombinant Escherichia coli have been prepared according to the industrial technology of Bacillus intermedius ribonuclease purification. Electrophoretic parameters and ultraviolet spectra characterize these preparations as homogeneous proteins. Immunochemical test system of identification of inactive RNAse for its purification has been worked out.     

Isolation of mRNA from protoplasts of spore-forming bacteria and study of its translation products

Total RNA was isolated from the protoplasts of Bacillus intermedius 7P which actively produced alkaline exocellular RNAse. A fraction of polyadenylated RNA was isolated from this RNA using chromatography on poly(U) Sepharose. The total RNA and poly(A) +RNA were translated in Xenopus laevis oocytes. Bacterial exocellular RNAse was identified among the products of translation by means of electrophoretic and immunological analyses. The mRNA of RNAse secreted by B. intermedius 7P was concluded to be polyadenylated.     

Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation

The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.     

The effect of Bacillus intermedius RNAse on the multiplication of Candida tropicalis yeasts]

The effect of Bacillus intermedius RNAse on the reproduction of Candida tropicalis and synthesis of the main biopolymers in the yeast cells. It has been found that stimulating action of the enzyme appears at the concentration of 10(-5)-10(-6) mg/ml and does not depend on the physiological state of the sowing culture. The connection between the increase of the ionic penetration and stimulation of the RNA and proteins synthesis in the yeast cells subjected to the RNAse action is shown. The mechanism of chromatine-associated RNA-polymerase activation is suggested to include the alteration of the ionic penetration of cells under the RNAse action.     

The cytomorphological evaluation of the effect of Bacillus intermedius RNAse on the organs of experimental animals]

Cytomorphological changes in organs of experimental animals after RNAse Bacillus intermedius treatment have been studied. It has been found that RNAse stimulates elements of lympho- and homopies of thymus and spleen and increases stromal reaction and functional activity of liver. The caused effects don't depend on catalytic enzyme activity.     

The histological evaluation of the effect of Bacillus intermedius RNAse on the organs of immunogenesis in laboratory animals]

Histological changes in organs of immunogenesis of experimental animals after RNAse Bacillus intermedius treatment have been studied. The effect of RNAse on organs is revealed in activation of both cell and humoral immunity and factors of nonspecific resistance at early stage of reaction. The inhibition of immunological reactions and factors of nonspecific resistance takes place at later stages of enzyme action. The caused effects don't depend on catalytic enzyme activity.     

Localization of alkaline phosphatase in cells of Bacillus intermedius

Alkaline phosphatase, an enzyme secreted by Bacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth of B. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na2HPO4 at a concentration of 0.01% diminished the activity of membrane-bound and extracellular phosphatases. CoCl2 at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

Extracellular ribonuclease from Bacillus pumilus]

The extracellular ribonuclease (RNAse Bp) was isolated from the cultural medium filtrate of Bacillus pumilus by ammonium sulfate precipitation and two stages of ion-exchange chromatography on carboxymethyl- and phospho-cellulose columns. The amino acid composition and N-terminal amino acid residue have been determined. The kinetic parameters of cleavage reaction of synthetic polynucleotides have been measured. According to their structural homology RNAse Bp has been shown to be similar to RNAses Ba and Bi. Catalytic properties of the enzyme are very close to RNAse Bi.     

Binase possesses a selective cytotoxic action on kit-transformed precursors of myeloid cells

The effect of cationic microbial ribonuclease from Bacillus intermedius (binase) on normal precursors of myeloid cells of FDC-P1 mice and kit-transformed precursors expressing the receptor of the growth factor of stem cells has been studied by flow-through cytometry. Selective apoptogenic properties of binase toward kit-transformed cells were revealed. Viable kit-transformed cells responded to binase by an increase in the concentration of cytosolic calcium. The content of calcium in the cytosol of both cell types in which apoptosis was induced by binase decreased in a dose-dependent manner. The death of cells was not accompanied by a substantial decrease in the content of intracellular RNA. A possible mechanism of binase-induced effects, which involves changes in the expression of genes due to the interference of exogenous RNAse into the RNA interference, was considered.     

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.     

The cloning and expression of the RNAse structural gene of Bacillus intermedius]

The structure gene of extracellular alkaline ribonuclease Bacillus intermedius (binase) has been cloned in E. coli cells in composition of pMT 316 plasmid carrying the inhibitor gene (barstar of barnase--binase structure homologue. The possibility to use such vector has been proved during the barstar action on binase catalytic activity. Using biochemical immunochemical analysis the expression of binase gene in E. coli cells has been confirmed. The recombinant clone E. coli which contains both plasmids simultaneously--carrying gene for barster and for benase has been produced. The given vector is suggested to be used for cloning of inhibitor gene to obtain a viable producer of alkaline intracellular ribonuclease.     

The effect of carbon sources and mononucleotides on the production of extracellular alkaline phosphatase by Bacillus intermedius

The biosynthesis of extracellular alkaline phosphatase in the streptomycin-resistant strains Bacillus intermedius S3-19 and S7 in the presence in the medium of 5'-nucleoside monophosphates and different sources of carbon--glucose, sodium pyruvate, sodium lactate, or glycerol--was studied. It was established that, in the presence of mononucleotides, the content of extracellular alkaline phosphatase in both strains increased; the maximal effect was caused by 5'-AMP at a concentration of 20 micrograms/ml. In medium with a low orthophosphate content, where active biosynthesis of alkaline phosphatase occurred, 1% glucose and 0.5% pyruvate stimulated this process 2.5-4 times, and 2% sodium lactate and sodium pyruvate, on the contrary, inhibited it by 20-40%. Analysis of the dynamics of growth and accumulation of extracellular phosphatase in the presence of different sources of carbon in the medium gives evidence of an interrelationship between the biosynthesis of alkaline phosphatase and carbon metabolism in Bacillus intermedius.     

The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis

Barnase, an extracellular RNAse from Bacillus amyloliquefaciens is secreted post-translationally from B. subtilis. The rate of secretion of barnase from B. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. However, the barnase signal peptide exported Escherichia coli alkaline phosphatase faster than mature barnase. Heat shock of B. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. The slow rate of export of barnase from B. subtilis is due to both the signal peptide and the mature protein sequence rather than either alone.     

Ribonuclease of Bacillus intermedius 7 P. Purification by chromatography on phosphocellulose and several characteristics of the homogeneous enzyme]

A new procedure for isolation of homogenous ribonuclease of Bac. intermedius from a commercial source is described. The yields of 140 mg of RNAse from 200 g of the enzymic powder were attained. The amino acid composition of the enzyme was determined. The RNAse contains neither the sulfhydryl groups nor the disulfide bonds and has only one histidine residue. At the same time the amount of aromatic amino acid residues is relatively high. The enzyme is highly resistant to heat and acid treatment but is less stable in an alkaline solution. The pH optimum of the RNAse for the RNA digestion is 8,5; the temperature optimum for this reaction is 37 degrees. A spectrophotometric method for the RNAse activity assay using polyA as a specific substrate was developed. The purified product provides a suitable starting material for structural studies.     

The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis

Barnase, an extracellular RNAse from Bacillus amyloliquefaciens is secreted post-translationally from B. subtilis. The rate of secretion of barnase from B. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. However, the barnase signal peptide exported Escherichia coli alkaline phosphatase faster than mature barnase. Heat shock of B. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. The slow rate of export of barnase from B. subtilis is due to both the signal peptide and the mature protein sequence rather than either alone.     

Hydrolytic enzymes and spore formation in Bacillus intermedius

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II-IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed.     

Membrane-bound forms of serine proteases of Bacillus intermedius

Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes.     

The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

Isolation and characterization of two alkaline ribonucleases from calf serum.

Treatment of calf serum at 60 degrees C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAse activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25 M KCl with a 6700-fold purification. The RNAse eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAse A serum and by the endogenous RNAse inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl2. This enzyme seems to be similar or identical to RNAse A. The other RNAse, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAse A or 5 mM MgCl2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3' -CMP and 2'- or 3' -UMP. Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate.