[Screening of potential antibiotics, inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis--2-C-methyl-D-erythritol-2,4-cyclodiphosphate derivatives].

The recently discovered nonmevalonate pathway of isoprenoid biosynthesis is a prospective target in screening of new antibiotics. Because of the absence of the pathway in the animal cells, the specific inhibitors of the pathway will be a new class of antibiotics against many pathogens (which cause, e.g., malaria, tuberculosis, etc), combining high efficiency and low toxicity. Several derivatives of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEC) were synthesized. 4-Phospho-methyl-D-erythritol-1,2-cyclophosphate, benzyl ether and benzyliden derivative of MEC inhibited the 14C-MEC incorporation into isoprenoids of chromoplasts from red pepper with IC50 of 1.7-5 MM. Some inhibition (about 10%) was also observed with the use of dimethyl ether and isopropyliden derivative of MEC.

[2-(14)C-Methylerythritol 2,4-cyclodiphosphate incorporation into bacterial and plant isoprenoids].

To work out a test system for studying the inhibition of isoprenoid biosynthesis through non-mevalonate pathway, the conversion of 2-(14)C-methylerythritol 2,4-cyclodiphosphate (14C-MEC) into isoprenoids of three bacteria species and into plastids of 11 higher plant species was studied. The highest efficiency (up to 63%) of the process was observed with chromoplasts from narcissus (Narcissus pseudonarcissus L.) and celandine (Chelidonium majus L.). Twenty five percent of added 14C-MEC was recovered in an isoprenoid fraction of red pepper (Capsicum annuum L.) chromoplasts. Thus, these three models can be used to test antibiotic inhibitors of isoprenoid biosynthesis.

[The role of 2-C-methylerythritol-2,4-cyclopyrophosphate in the resuscitation of the "nonculturable" forms of Mycobacterium smegmatis].

2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate (MEC), an intermediate of the biosynthesis of isoprenoid compounds in bacteria, was found to be capable of exerting a resuscitating effect on resting Mycobacterium smegmatis cells. The introduction of an additional copy of the ispE gene encoding cytidyl-methylerythritol kinase, an enzyme involved in MEC synthesis in M. smegmatis, resulted in the emergence of a capacity for spontaneous reactivation of "nonculturable" M. smegmatis cells, which is not characteristic of the wild-type cells of this species. The involvement of MEC in the transition from the "nonculturable" state to the state of active growth is indicative of a previously unknown function of MEC, assumed to consist in regulation of the bacterial genome activity.

[2-C-Methylerythritol phosphate pathway of isoprenoid biosynthesis as a target in identifying of new antibiotics, herbicides, and immunomodulators (Review) ].

Specific inhibitors of 2-C-methylerythritol phosphate pathway (MEP-pathway), including compounds obtained based on its metabolites, may compose a new class of antibiotics combining high efficiency and low toxicity. MEP-pathway of isoprenoid biosynthesis is a promising target in identifying new herbicides, immunomodulators, and other physiologically active compounds.

[The character of the interaction of beta-carotene-15,15'-dioxygenase from rabbit small intestine with lycopene, 15,15'-dehydro-beta-carotene, lutein, and astaxanthine]. / O kharaktere vzaimodeistviia beta-karotin-15,15'-dioksigenazy iz tonkogo kishechnika krolika s likopinom, 15,15'-degidro-beta-karotinom, liuteinom i astaksantinom.

Interaction of rabbit small intestine mucosa beta-carotene 15,15'-dioxygenase with lycopene, 15,15'-dehydro-beta-carotene, lutein and astaxanthine is unaccompanied by the formation of products of enzymatic cleavage of the central double bond of these carotenoid molecules. Apo-carotenoids of lutein and lycopene are formed by nonenzymatic oxidation. Lycopene and 15,15'-dehydro-beta-carotene competitively inhibit the enzymatic oxidation of beta-carotene. Inhibition of the enzyme conversion by lutein and astaxanthin is less strong, being of a competitive-noncompetitive type. Enzymatic formation of retinal from beta-carotene depends on the accessibility of substrate molecules.

[Properties of beta-carotene-15,15'-dioxygenase, stabilized during purification with lutein and dithiothreitol]. / Svoistva beta-karotin-15,15'-dioksigenazy, stabilirzirovannoi v protsesse vydeleniia liutenom i ditiotreitolom.

Preparations of beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) obtained with or without lutein and dithiothreitol (DTT) as stabilizers display similar properties. The stabilized preparation has an apparent Km of 17 microM and Vmax of 2.8 nM/h/mg of protein, a pH optimum of 8.1, is sensitive to SH-agents and is activated by Fe2+, DTT or glutathione used at optimal concentrations of 0.5 mM, 2 mM, and 5 mM respectively. At DTT concentration above 2 mM the enzyme activity decreases drastically, which testifies to the importance of maintaining the thiol and disulphide group ratio in the protein molecule at an optimal level. At 4 mM Fe2+ there occurs a nonenzymatic formation of apo-carotenals; however, retinal is formed only after addition of commensurate amounts of thiol compounds. The enzyme activity depends on the ionic strength of the incubation medium, the maximal effect being observed with 0.4 M K-phosphate buffer. Substitution of phosphate for Cl- partly inhibits the enzyme. The optimal temperature for this reaction is 45 degrees. A simultaneous use of optimal incubation parameters (2 mM DTT, 0.4 M K-phosphate buffer pH 8.1 and 45 degrees C) increases the retinal yield 2.5-fold against control.

[Enzymatic conversion of torulene and torularhodine into retinal]. / Fermentativnoe prevrashchenie torulina i torularodina v retinal'.

The enzymatic conversion of torulene to retinal is demonstrated in in vitro tests using beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) from the rabbit small intestine mucosa. This is the first evidence for the potent vitamin A activity of torulene. Little amounts of retinal also formed from methyl ester of torularhodin and from torularhodin.

[Stabilization and protection of an enzymatic system, participating in the transformation of beta-carotene into retinal, during its isolation]. / Stabilizatsiia i zashchita fermentnoi sistemy, uchastvuiushchei v orevrasgcgebuu v beta-karotina v retinal', pri ee vydelenii.

The authors studied the effect of dithiothreitol (DTT), carotenoids and protease inhibitors on stabilization and protection of the enzyme catalysing the conversion of beta-carotene into retinal during the enzyme isolation from the rabbit small intestine. The addition of 1 mM DTT into the homogenization mixture increased the activity of the enzyme 5 times compared with control. The additional introduction of 0.7 mg/ml soybean trypsin inhibitor or 2.10(-4) M phenylmethylsulfonyl fluoride increased the enzyme activity 2.1 and 1.2 times, respectively. Lutein, beta-carotene and lycopene at a concentration of 10 mg/ml increased the enzyme activity 2.1, 1.9 and 1.6 times respectively. The effects of DTT, lutein and the protease inhibitor depended on their concentrations and was of an independent additive character. The maximum activity of the isolated enzyme exceeded the control without DTT 15 times.

[Cloning and detailed mapping of the fra-ymt region of the Yersinia pestis pFra plasmid]. / Klonirovanie i detal'noe kartirovanie fra-ymt-oblasti plazmidy pFra Yersinia pestis.

The genetical libraries of the pFra plasmid of Yersinia pestis genes were obtained by insertion into the PstI, SalGI, EcoRI, XhoI restriction sites of the cosmid vector pHC79. The immunochemical analysis of the recombinant clones has revealed the clones synthesizing the antigen Fl (fraction I) and mouse toxin (Ymt--Yersinia pestis murine toxin). The restriction analysis of the plasmids from antigen synthesizing clones has permitted to construct the detailed physical map of the fra-ymt region of the pFra plasmid the size of 22 kb. The recombinant F1 positive clones of Escherichia coli are able to form at 37 degrees C the capsule-like structure peculiar for Yersinia pestis. The antigen F1 and the mouse toxin were isolated, purified and characterized. The antigen F1 is an 1-2 Md polymer containing a 16 kDa protein subunit. The mouse toxin a 240 kDa protein consisting of 61 kDa subunits. The nucleotide sequence of ymt gene has been defined.

[Behavior of Sa plasmid in tularemia pathogen cells]. / Povedenie plazmidy Sa v kletkakh tuliaremiinogo mikroba.

The genome of Sa plasmid is shown to be a subject of genetical rearrangements in Francisella tularensis cells. The rearrangements either result in plasmid integration into the host cell genome or intramolecular amplification of cat-gene with the subsequent excision and recombination of the derivative plasmids. Stable inheritance of the plasmid is registered after integration while plasmid elimination occurs in case of extrachromosomal localisation.

[Features of the interaction of Escherichia coli and Francisella tularensis RNA polymerases with hybrid plasmids bearing fragments of Francisella tularensis chromosomal DNA]. / Osobennosti vzaimodeistviia RNK-polimeraz Escherichia coli i Francisella tularensis s gibridnymi plazmidami, nesushchimi fragmenty khromosomnoi DNK Francisella tularensis.

Hybrid plasmids containing the fragments of Francisella tularensis chromosomal DNA and capable of tet-gene expression both in Escherichia coli and Francisella tularensis cells were constructed. The regions of francisella chromosomal DNA binding the RNA-polymerases of Escherichia coli and Francisella tularensis were found by the electron microscopy technique. Interconnection of those regions with the expression of tet-gene of the hybrid plasmids was demonstrated.

[Characteristics of non-nucleolar RNP structures in hepatocyte nuclei in the early stages of the stimulation of proliferation]. / Kharakteristika neiadryshkovykh RNP-struktur iader gepatotsitov na rannikh stadiiakh stimuliatsii proliferatsii.

Quantitative changes of extranucleolar RNP-structures were studied on ultrathin cell sections of hepatocytes in the first hours after partial hepatectomy of guinea pigs. This purpose in mind, the sections were treated according to Bernhard. In 2.5 hours after the operation, the relation quantity of perichromatin and interchromatin fibrils increased, whereas the number of perichromatin and interchromatin granules decreased. Therefore the early response of hepatocytes to the proliferative stimulus represents not only intensification of the synthesis of a newly formed hnRNA but also enhancement of the processing and transport of this RNA. After 5 hours the relative quantity of both types of RNP-fibrils diminished to the control level, while the number of perichromatin granules was even smaller than that in the previous case. This points out that the transcription of the extranucleolar RNA becomes less active, and the processing and transport of the earlier synthetized RNA continues. Within the next time lapse (9 hours) the relative quantity of all the studied RNP-structures was low, the content of perichromatin granules being dramatically decreased as compared to that after 5 hours. A comparison of changes in RNP-structures with the degree of condensation of chromatin allows a conclusion that the decondensation of some part of condensed chromatin after 2.5 and 9 hours is functionally different. If initially it is required for the intensification of the template synthesis, thereafter it may be connected with the preparation for replication.

[Recovery of activity of the gene coding for tetracycline resistance in the plasmin pBRS188]. / Vosstanovlenie aktivnosti gena ustoichivosti k tetratsiklinu plazmidypBRS188.

The spontaneous recovery of activity of tet gene deleted of the promoter region was studied. Plasmid pBRS188 was used as a model for studying this problem. The plasmid has the fragment of tet gene of pBR322, from which it originates, between the sites of restriction endonucleases EcoRI and HindIII cleavage resulting in inactivation of tet promoter. E. coli cells harbouring the plasmid were shown to revert the TcR phenotype with the frequency 10(-9). The gene activation coincided with intraplasmid recombination revealed by restriction analysis. In some cases the recovery of tet gene activity coincided with the formation of multimeric plasmids.

[Electron microscope study of chromatin in hepatocyte nuclei during the first hours after partial hepatectomy. V. Changes in the relative area of condensed chromatin and the density of chromatin fibril packing in the ultrathin sections]. / Elektronno-mikroskopicheskoe izuchenie khromatina v iadrakh gepatotsitov v pervye chasy posle chastichnoi gepatéktomii. V. Izmenenie otnositel'noi ploshchadi kondensirovannogo khromatina i plotnosti upakovki fibrill na ul'tratonkikh srezakh.

The degree of chromatin condensation was studied on ultrathin cell sections of guinea pig hepatocytes during the prereplicative period after partial hepatectomy. Three time points were chosen for analysis namely 2,5, 5 and 9 hrs after operation since they show marked increasing (2.5 hrs), decreasing (5 hrs) and repeated increasing (9 hrs) of the amount of ethidium bromide binding to chromatin. The degree of chromatin condensation was determined by measuring the area occupied by condensed chromatin and also by measuring the number of chromatin fibrils per a certain length. The condensed chromatin with varying localization in the nucleus were studied separately. The changes of nucleoplasmic chromatin were most pronounced: at 2.5 and 9 hrs after operation the decrease of the relative area and of the density of chromatin fibrils package was observed; these parameters were near to control at 5 hrs after operation. In general the changes in nucleoplasmic chromatin were correlated with the changes of the activity of the chromatin in the whole nucleus. The decondensation of the perimembranous chromatin was manifested in the decrease of its area and was expressed only at 9 hrs after operation. The perinucleolar chromatin was found to show the gradual decondensation which was manifested mainly by the decrease of its relative area. Thus the condensed chromatin seems to be a labile structure which undergoes essential changes in the process of the exit of the hepatocytes from G0-stage of the cell cycle, during the prereplicative period.

[Electron microscopic study of the chromatin in hepatocyte nuclei in the 1st hours after a partial hepatectomy. IV. Increased sensitivity of active chromatin to EDTA exposure]. / Elektronno-mikroskopicheskoe izuchenie khromatina iader gepatotsitov v pervye chasy posle chastichnoi gepatéktomii. IV. Povyshennaia chuvstvitel'nost' aktivnogo khromatina k vozdeistviiu EDTA.

In the period of increasing the guinea-pig hepatocyte chromatin template activity, 2.5 hours after partial hepatectomy, an increased susceptibility of condensed chromatin to the bleaching action of EDTA in the Bernard reaction has been found. The condensed chromatin of the activated by partial hepatectomy guinea-pig hepatocytes, studied on ultrathin sections, is bleached under the action of EDTA more intensely compared to the chromatin of the control (non-activated) cells. Five hours after partial hepatectomy, when hepatocyte chromatin, according to its physico-chemical properties and functional activity, is the same as that of the control (non-operated) animals, its capacity of being bleached by EDTA also returns to the control level. In one nucleus studied on ultrathin sections the perinucleolar chromatin was found to be more sensitive to EDTA than the chromatin of other parts of the nucleus. It is suggested that the treatment with EDTA under given conditions can be used for revealing the functional state of chromatin on ultrathin sections.

[Electron microscope examination of chromatin in hepatocyte nuclei within the first hourse after partial hepatectomy, II. The degree of chromatin condensation and the organization of fibrillar RNP components]. / Elektronno-mikroskopicheskoe izuchenie khromatina v iadrakh gepatotsitov v pervye chasy posle chastichnoi gepatektomii. II. Stepen' kondensatsii khromatina i organizatsiia fibrilliarnykh RNP-komponentov.

The state of hepatocyte chromatin (the area occupied by the regions of condensed chromatin on ultrathin sections and the quantity of perichromatin RNP fibrils which was estimated by the area of the fibrillar zone and the concentration of fibrils within the same zone) were studied within the first hours after partial hepatectomy of guinea pigs. The area occupied by the regions of condensed chromatin on preparations with differentially revealed DNP and RNP components decreased by 12% in 2.5 hours since the operation had been performed, became normal in 5 hours, and again decreased by 30% in 9 hours. Decondensation of chromatin was accompanied with the increase of the number of perichromatin RNP fibrils, products of template activity of chromatin, and the rise of ethidium bromide binding. The binding of ethidium bromide by the chromatin of hepatocytes increased by 39% in 2.5 hours, returned to the control level in 5 hours and again increased by 22% in 9 hours.