[Prevalence of nonmevalonate and mevalonate pathways for isoprenoid biosynthesis among bacteria of different systematic groups].

The effect of fosmidomycin and mevinoline, inhibitors of the nonmevalonate and the mevalonate pathway of isoprenoid biosynthesis, respectively, on the growth of 34 anaerobic and 10 aerobic prokaryotic strains was studied. Fosmidomycin at the concentrations used was shown to inhibit the growth of 9 (of 10) representatives of the family Microbacteriaceae, 4 (of 5) strains of Thermoanaerobacter, and 11 (of 12) strains of Clostridium, whereas mevinoline inhibited the growth of lactobacilli (Carnobacterium), methanogenic and sulfate-reducing bacteria insensitive to fosmidomycin. During the late growth phase, four strains of actinobacteria (of nine) accumulate the compound, which, upon oxidation, generates a long-lived free radical; three strains synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC). It was concluded that the difference in the sensitivity of the organisms to fosmidomycin and mevinoline might serve as a test to differentiate several representatives of the family Microbacteriaceae. The use of mevinoline for inhibiting methanogens in ecological investigations seems to be promising.

[Free radicals in mercury-resistant bacteria indicate a novel metabolic pathway]. / Svobodnye radikaly u rtut'-rezistentnykh bakterii kak indikatory novogo metabolicheskogo puti.

A mercury resistant-soil bacterium P.10.15, identified as a close relative of Pseudomonas veronii, was shown to accumulate a specific compound in the stationary phase of growth. This compound is converted to a long-lived free radical under oxidizing conditions, as registered by its EPR signal at room temperature. The compound was purified by ion-exchange and gel-filtration chromatography and identified by mass spectroscopy, 2D NMR, and EPR as a trisaccharide beta-D-GlcpNOH,CH3-(1-->6)-alpha-D-Glcp-(1-->1)-alpha-D-Glcp, or, in other words, as 6-O-(2-deoxy-2-[N-methyl]hydroxylamino-beta-D- glucopyranosyl)-alpha-alpha-trehalose, previously discovered in Micrococcus luteus (lysodeikticus) and named lysodektose. The compound is suggested to be a novel intermediate of a previously unknown basic metabolic pathway of trehalose transformation in bacteria, a potential target for antibacterial drug development.

[Effect of fosfidomycin on development of various infections in mice]. / Deistvie fosmidomitsina na razvitie nekotorykh infektsii u myshei.

Antibiotic fosmidomycin will know as inhibitor of the nonmevalonate pathway of isoprenoid biosynthesis and as possible antimalarial drug, was shown to possess a certain protective effect on mice experimentally infected with tularemia, tiphus or coli-septicemia. Positive effect on mice with chronic form of tuberculosis was not observed when the animals were given 1 mg of fosmidomycin per capita twice a day. Under oxidative conditions an ESR signal of long living nitroxil free radicals were registered in the water solution of fosmidomycin. The radicals are supposed to be involved in the therapeutic effect of the antibiotic.

Comparative characterization of methods for removal of Cu(II) from the active sites of fungal laccases.

Copper-containing sites of laccases isolated from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus were characterized by optical methods and EPR spectroscopy. Methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. The data of EPR spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 copper. The original enzymes can be reconstituted from the derivatives by dialysis under anaerobic conditions, resulting in complete recovery of native conformation of the protein molecule and the structure of the copper-containing site.

[Changes in the EPR spectra of the nitrosyl complexes of blood proteins in the low-intensity whole-body irradiation of mice]. / Izmenenie spektrov EPR nitrozil'nykh kompleksov gemoproteinov krovi pri nizkointensivnom total'nom obluchenii myshei.

After NO adding to mice blood and isolated erythrocytes ESR signal of nitrozyl complex HbNO (g = 2.07, g = 1.98) and NO-induced MetNg (g = 6.0) were registered. It was shown that the intensity of ESR spectra of these complexes increased after radiation of mice with a dose of 0.06, 0.6 and 5.4 cGy. Low-dose irradiation (0.6 and 0.06 cGy) caused the change in the form of ESR spectra of HbNO (g = 2.07), which is indicative of the shift from T-structure to R-structure and of the preferred formation of R-conformations of oxyhemoglobin in blood. It was found that dependence of NO-induced MetHb signal on irradiation dose is bimodal that may be connected with nonlinear response of the cells to irradiation and retarded adaptive response after radiation with low doses.

[Interaction of bacteria with mercuric compounds]. / Vzaimodeistvie bakterii s rtutnymi soedineniiami.

We investigated the interaction of mercuric compounds with the bacteria Corynebacterium ammoniagenes, Micrococcus luteus, and Mycobacterium smegmatus capable of producing hydroxylamines (R-NOH) and 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MECP), which are prone to form free radicals. The interaction of these substances with Hg2+ ions and their dynamics during the mercuric poisoning of bacteria was studied by EPR and NMR. Under stress conditions induced by lowering pH or generation of active oxygen species, the bacteria and, especially, their mutants with enhanced sensitivity to oxidative stress, were found to respond to exposure to 1-3 micrograms/ml HgCl2 and p-chloromercuribenzoate by a several-fold increase in their viability. The data obtained were interpreted in terms of the involvement of the sulfhydryl groups of bacterial surface proteins in this phenomenon. The interaction of bacteria with mercuric compounds may affect the pathogenesis of tuberculosis and other diseases.

Laccase of Coriolus zonatus: isolation, purification, and some physicochemical properties.

Laccase is one of the lignolytic enzymes found in liquid cultures of the fungus Coriolus zonatus in defined medium. The enzyme was isolated from culture liquid and characterized. Laccase from C. zonatus is a single-chain protein with a molecular mass of 60 kDa. Carbohydrate moiety of enzyme consisted of mannose, galactose and N-acetyl-glucosamine in a ratio of 6:2:0.6, respectively, and comprised 10% of the entire molecule. Isoelectric point was detected at pH 4.6. Laccase was found to have a pH optimum of 4.9 and temperature optimum of 55 degrees C. Substrate specificity studies were conducted with catechol, K-ferrocyanide, hydroquinone, and sinapinic acid as substrates. The highest efficiency of catalysis was observed with sinapic acid as the substrate. The kinetic constants kcat and Km of this reaction were 624 s-1 and 7 microM, respectively.

[EPR-spectroscopy of human blood preparations. Remote sequelae of the Chernobyl accident]. / EPR-spektroskopiia preparatov krovi cheloveka. Otdalennye posledstviia Chernobyl'skoi avarii.

It was shown, that as a result of Chernobyl accident the intensity of the EPR-signal of hemoproteins and ferroporphyrins of people blood increased, that most probably connected with disturbing of ferroprotein metabolism. Maximum intensity values had people, irradiated with low doses (to 2 cGy), that corroborate the theory of danger these doses for the people organism.

[Inhibition of endogenous oxidation of DNA by spin traps]. / Ingibirovanie éndogennogo okisleniia DNK spinovymi lovushkami.

It was shown that spin trap feeding resulted in a decrease of oxidation of rat DNA. This is an evidence of free radical mechanism of endogenous DNA oxidation.

[The effect of structure of benzimidazoles on the character of forming intramolecular cross-links in DNA and chromatin]. / Vliianie struktury preparatov klassa benzimidazola na kharakter obrazovaniia vnutrimolekuliarnykh sshivok v DNK i khromatine.

An investigation of a number of benzimidazole class preparations, being distinguished by a position of aminomethyl substitutes, has been carried out. It has been shown, that the non-substituted preparation BIO-10 does not form UV-cross-links in DNA and chromatine; BIO-40, having one substitute in the position 2, causes the formation of inter-molecular cross-links DNA-DNA. The preparation BIO-50, having 2 aminomethyl groups in the imidazole nucleus positions 2 and 6, forms cross-links DNA-DNA and DNA-protein in chromatine. The generation of radicals by the preparations BIO-10 and BIO-50 has been studied by the EPR-method by use of spin trap. It has been demonstrated, that BIO-10, unlike BIO-50, actively generates superoxide. A supposition has been made, that an UV-formation of superoxide-radical in the presence of BIO-10 might be a reason of DNA-macromolecule destruction.

[Titration of SH-groups of histone H3 in a DNP preparation of normal and neoplastic animal cells with a mercury-containing spin marker and with a DTNB preparation]. / Titrovanie SH-grupp gistona N3 v DNP normal'nykh i opukholevykh kletok zhivotnykh rtut'soderzhashchei spinovoi metkoi i preparatom DTNB.

DNP samples isolated from the cells of calf thymus and Ehrlich ascite carcinoma of mice were examined. SH-groups of histone H3 of chromatin from these cells were titrated with mercury-containing spin label and with DTNB under joint action of different salt and sarcosyl concentrations on DNP. The results revealed differences in accessibility and titration of histone H3 SH-groups in DNP of normal and tumor cells with DTNB, as well as in molecular dynamics of the mercury-containing spin label introduced to these SH-groups.

[The action of benzimidazole derivative preparations on the formation of DNA-protein cross-links in the UV irradiation of chromatin]. / Deistvie preparatov proizvodnykh benzimidazola na obrazovanie sshivok DNK--belok pri UF-obluchenii khromatina.

Effect of benzimidazole-derivatives on the DNA-protein binding formation was studied after UV-radiation of chromatin. These derivatives were shown to protect chromatin from UV-induced DNA-protein binding formation. Structural analog contained two aminomethyl residuals sensibilized additional binding formation in chromatin. Results suggested, that benzimidazole interacted with DNA, while aminomethyl groups interacted with protein and sensibilized binding of DNA with histone H1.

Lysodektose--a new trisaccharide from Micrococcus lysodeikticus which is transformed into an aminoxyl free radical with the loss of an electron.

A new trisaccharide 6-0-(2-deoxy-2-(N-methyl)-hydroxylamino-beta-D- glucopyranosyl)-alpha,alpha-trehalose named lysodektose has been isolated from Micrococcus lysodeikticus. In oxygenated solutions or in the presence of K3 Fe (CN)6 lysodektose is transformed into a long lived free radical. Spin trapping data are presented and functions are suggested for the substance.

Derivatives of glutamic acid and trehalose which can be transformed into long-living free radicals by the loss of an electron are identified in some bacteria.

A derivative of glutamic acid (ammonigenin) and a trisaccharide named lysodektose which are converted into long-living free radicals by the loss of one electron were isolated from Brevibacterium ammoniagenes and Micrococcus lysodeikticus. Structural formulae suggested for both substances based on ESR-, NMR- and mass spectra, isotopic substitution experiments and other data are: lactone of N-hydroxy-N-(2-carbamoylethyl)-glutamyl-4-amino-2-hydroxybutyric amide and 6-O-[2-deoxy-2-(N-methyl)-hydroxylamino-beta-D-glucopyranosyl]- alpha, alpha-trehalose. Radical forms appear on reversible oxidation of hydroxylamino groups to nitroxyl groups. Participation in the protection of bacterial cells and regulation of their metabolism is suggested for these compounds.

[UV-induced formation of DNA-protein cross-links in chromatin in the presence of chemical agents]. / UF-indutsirovannoe obrazovanie sshivok DNK-belok v khromatine v prisutstvii khimicheskikh agentov.

Hindered phenols, quinones, and SH-compounds have been studied as possible protectors and sensitizers of DNA-protein cross-linking in chromatin. Efficacy of cross-linking was estimated by the quantity of protein linked to DNA after UV irradiation at 254 um in the presence of chemical agents. Phenols and quinones exert protective influence on cross-linking whereas DNA-protein cross-links are sensibilized by cysteine hydrochloride.

[Local homology of amino acid sequences of histones H3, H4 and the protein repressor lambda Cro. Possible conformation of homologous histone sites in DNP]. / Lokal'naia gomologiia aminokislotnykh posledovatel'nostei gistonov H3, H4 i belka repressora lambda Cro. Vozmozhnaia konformatsiia gomologichnykh uchastkov gistonov v DNP.

A mathematical analysis of amino acid sequences was carried out with a view of detecting possible homology between histones H3 and H4 and repressor-activator proteins of prokaryotes according to the A. I. criterion which reflects the similarity of their primary structure. It was found that the sites of eukaryotic histones H3 (102-123) and H4 (68-85) and site alpha 3 (24-25) of the prokaryotic repressor protein lambda Cro, i. e., the site of protein interaction with DNA, reveal a statistically significant homology. The A. I. value for the H3 site of lambda Cro is 3.37, that for the H4 site of calf thymus and sea horse is 3.28. The amino acid sequences of these proteins in the alpha 2-alpha 3 site, i. e., the site in which the homology between amino acid sequences of histones and DNA-binding proteins had been established previously, with regard to similarity of their secondary structure of the helix-turn-helix type, were analyzed. A pairwise comparison of H3 and protein lambda Cro showed that the A. I. value for histones H3 from various sources is approximately 2.7; however, the homology of the alpha 2 site is lower than that of site alpha 3. It is concluded that there exists an evolutionary relationship between homologous segments of histones H3 and H4 and protein lambda Cro, which can be preserved in order to maintain a definite secondary structure, presumably for binding to DNA.

[Study of a free radical compound from the cells of the bacteria Micrococcus lysodeikticus]. / Issledovanie svobodnoradikal'nogo soedineniia iz kletok bakterii Micrococcus lysodeikticus.

Some physico-chemical properties of a compound previously detected in M. lysodeikticus cells were studied. This compound is hydrophilic, has hydrophilic, has a low molecular weight and does not contain The redox potentials of the free radical formation in the cytoplasm and in purified preparations were determined. A hypothetical reaction scheme is proposed.

[Study of spin-labeled microsomal membrane proteins by their reaction with ascorbic acid]. / Izuchenie spin-mechennykh belkov membran mikrosom po reaktsii s askorbinovoi kislotoi.

Kinetics of the reduction of nitroxyl radicals bound to SH-groups of microsome membrane proteins by ascorbic acid was investigated. The reduction involves two steps. During the first quick step mainly the radicals with weak immobilization located on the membrane surface are reduced. At the second slow step more immobilized radicals located in hydrophobic membrane regions take part in the reaction. A kinetic analysis of the process showed that the slow step is not determined by the slow movement of protein or its fragments, for instance, from the membrane depth to its surface, and apparently is conditioned by lower ascorbic acid concentration in the membranes.