Kinetic model of hydrocortisone 1-en-dehydrogenation by Arthrobacter globiformis

A kinetic model of the hydrocortisone-to-prednisolone transformation by Arthrobacter globiformis is constructed using the experimental data obtained in studies of this process. Besides adequately describing experimental data, the model allows one to determine the relation between hydrocortisone oxidation and the level of endogenous substrates in bacterial cells, and the relation between the saturating concentration of hydrocortisone in the enzymic system of bacteria and the content of endogenous substrates in their cells, as well as the regulation of the transmembrane potential and the activation by the uncouplers.     

The physiologic role of pyocyanine synthesized by Pseudomonas aeruginosa

The physiological role of pyocyanine for Pseudomonas aeruginosa was studied. Its synthesis was shown to commence at the retardation growth phase. Pyocyanine was accumulated only in the growth medium. The addition of 2,6-dichlorophenolindophenol accepting the reducing equivalents from coenzyme Q and transferring them to cytochrome c inhibited the pigment accumulation. This was indicative of the connection between pyocyanine synthesis and the level of the reducing equivalents in the cells. Pyocyanine did not accept the reducing equivalents from coenzyme Q in the respiratory chain of P. aeruginosa. Only reduced pyridine nucleotides served as substrates for pyocyanine in the reaction of autooxidation. The kinetic parameters of this reaction and the affinity of NADH dehydrogenase for the substrate were measured. The kinetic data were analysed to show that, under the physiological conditions, pyocyanine could not apparently compete with the respiratory chain for the reducing equivalents and hence directly regulate the level of NAD(P)H in P. aeruginosa cells. In order to keep the oxidising activity at a level necessary for the cells, the latter decreased the content of the reducing equivalents either by synthesizing pyocyanine or owing to the activity of cyanide-resistant oxidase. These processes of releasing the reducing equivalents are in a reciprocal relationship.     

Effect of limitation of bacterial growth with carbon, sulfur and iron on the synthesis of cytochromes, development of cyanide-resistant respiration and super-synthesis of metabolites

The work is concerned with the effect produced by limiting the growth of various bacteria with carbon, sulfur and iron on cytochrome synthesis, development of cyanide-resistant respiration and oversynthesis of metabolites. The cessation of bacterial growth due to the exhaustion of a carbon source was shown to be accompanied with the development of cyanide-resistant respiration though the oversynthesis of metabolites did not occur. If the growth was limited by a sulfur or iron source, the concentration of cytochromes a, b and c fell down as compared with that when the growth was limited by a carbon source, and metabolites were produced and accumulated in the medium. In that case, the respiration of virtually all the bacteria was inhibited by cyanide to a great extent. As was demonstrated for Pseudomonas aeruginosa, the development of cyanide-resistant respiration was inhibited when metabolites accumulated and then the respiration became completely resistant to cyanide as soon as the oversynthesis ceased. Apparently, whatever limits the bacterial growth, the process of oversynthesis inhibits cyanide-resistant oxidase.     

A study of interactions of platinum (II) compounds with DNA by means of CD spectra of solutions and liquid crystalline microphases of DNA

The optical properties of the DNA complexes with divalent platinum compounds of the cis-diamine type differing both in the nature of anionic and neutral ligands and in the spatial arrangement about the platinum atom were studied. The platinum compounds cis-[Pt(NH3)2Cl2], [Pt(en)Cl2], [Pt(tetrameen)Cl2], cis-[Pt(NH3)2NO2Cl], and cis-[PtNH3(Bz)Cl2] at small values of r (r is the molar ratio of a platinum compound to DNA nucleotides in the reaction mixture) were found to induce an increase in the amplitude of the positive band in the circular dichroic (CD) spectrum of linear DNA. All the compounds listed except cis-[Pt(NH3)2NO2Cl] caused a sharp decrease of the amplitude of the negative band in the CD spectrum of a liquid crystalline microphase of DNA formed in solution in the presence of poly(ethylene glycol). All these platinum compounds (except [Pt(tetrameen)Cl2]) exhibit biological (antimitotic, antitumour, etc.) activity. The platinum compounds trans-[Pt(NH3)Cl2], trans-[Pt(NH3)2NO2Cl], cis-[PtNH3PyCl2], cis-[Pt(NH3)2(NO2)2], and [Pt(NH3)3Cl]Cl exhibiting a low (if any) biological activity, either induced a decrease of the amplitude of the positive band in the CD spectrum of linear DNA, or did not affect the CD spectrum at all. The effect of these platinum compounds on the CD spectrum of the liquid crystalline microphase of DNA was either weak or absent. It is assumed that the specific biological action of platinum compounds of the cis-diamine type is determined by the polydentate binding to DNA: in addition to the cis-bidentate covalent binding of platinum to DNA nitrogen bases, a hydrogen bond formation between the DNA and cis-amino ligands occurs by means of protons at nitrogen atoms.     

Effect of biologically active compounds of divalent platinum on the properties of the liquid crystal "microphase" of DNA

The optical properties of the "microphases" modeling the state of the DNA molecule in the cell and formed of both the low molecular DNA and the DNA complexes with cis- and trans-isomers of dichlorodiamine platininum (II) were studied. It was shown that the intensive band characteristic of the circular dichroism spectrum of the initial DNA "microphase" was decreasing with binding of DNA to cis-Pt (II) or trans-Pt (II). The effect of cis-Pt (II) on the "microphase" optical properties was more significant than that of trans-Pt (II). The effect correlated with the biological activity of the cis- and trans-compounds of platinum. Possible causes of the decrease in the optical activity of the DNA "microphase" are discussed.     

On the absence of correlation between cyanide-resistant respiration and cytochrome d content in bacteria

The regularity of appearance of cyanide-resistant respiration and cytochrome d in various bacteria as well as the relationship between the degree of resistance of respiration to cyanide and cytochrome d content was studied. Bacteria able to synthesize cyanide-resistant respiration were shown to appear during transition of culture to the stationary phase of growth caused by the exhaustion of carbon source. No regulatory of appearance of cytochrome d was observed. There is no correlation between the degree of resistance to cyanide and cytochrome d content. It was concluded that the cyanide-resistant respiration of bacteria and eukaryotic microorganisms may be associated with the functioning of a non-cytochrome nature oxidase.     

A compact from of methylated DNA is solutions containing poly (ethylene glycol).

Some peculiarities of compactization of double-stranded DNA molecules containing methylated nitrogen bases have been studied in water-salt solutions of PEG. It is shown that the methylation of N7-atoms of guanyl residues in original DNA molecules does not prevent the formation of DNA compact particles, but results in a decrease of the amplitude of the negative band in the CD spectrum of compact particles. The influence of N7-guanine methylation on the shape of the CD spectrum being the greater, the lower is the concentration of PEG. The dependence of the negative band amplitude in the CD spectrum on the content of methylated guanyl residues is practically the same for low-molecular weight DNA's from different sources. The observed decrease in the negative band amplitude is interpreted as a result of alterration of guanyl residue orientation relative to the helix axis which leads to diminished optical activity of the "microcrystalline" domains of compact particles. The evidence obtained suggests that changes in the secondary structure of DNA lead to considerable difference between CD spectra of compact particles of methlated DNA and psi-form of DNA. (The changes in the CD spectrum of the DNA compact particles occur also as a result of methylation of C5-atoms of cytosine residues). It is suggested that the negative band in the CD spectrum can be used a criterion for detection of negligible alterations in the DNA secondary structure.     

Cloning and expression of Clostridium thermocellum F7 cellulase genes in Escherichia coli and Bacillus subtilis cells

The Clostridium thermocellum total DNA Sau 3A fragments' library was constructed on the basis of shuttle vector pMK4 for the Escherichia coli - Bacillus subtilis. 14 clones with endoglucanase activity and one with beta-glucosidase activity were selected in E. coli cells. Recombinant plasmids pCE were characterized by structural instability of various degree in B. subtilis cells. The results of the physical mapping, analysis of gene products in E. coli mini-cells as well as the DNA-DNA blot hybridization have led to conclusion on cloning of 7 individual genes for endoglucanases. Up to 3 polypeptides of various molecular weight corresponding to the products of cel gene were revealed in E. coli mini-cells containing the recombinant plasmids. The hybridization analysis demonstrated considerable homology of the majority of cel genes.     

Reverse electron transfer from the cytochrome c level in cyanide-resistant mitochondria of the yeast, Candida lipolytica]

The ability of cyanide-resistant mitochondria of yeast Candida lipolytica to perform reverse electron transfer from cytochrome c to alternative oxidase was studied. It was shown that the energy for such a transfer can be provided by high energy intermediates or membrane potential but not by ATP. Reverse electron transfer from cytochrome c is impossible due to energy of NADH and alpha-glycerophosphate oxidation via alternative pathway in the presence of cyanide. These results prove once again that electron transfer via alternative pathway is not connected with the energy accumulation.