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Inhibition of mollicutes by synthetic oligonucleotides and their analogs complementary to specific "signature" regions of 16S rRNA and corresponding sequences of ribosomal operon DNA was studied. It was shown that antisignature oligonucleotides inhibited transcription in vitro for above 79% interacting specifically with ribosomal operon and non-specific with DNA-dependent RNA-polymerase. The inhibition efficiency depended on oligonucleotide sequence and type of modification. Translation in vitro was suppressed most efficiently (up to 60%) by oligonucleotides complementary to 3'-end region of 16S rRNA, also depending on their modification. Translation in vivo was inhibited most efficiently (up to 73%) by thiophosphate analogs of oligonucleotides complementary to sequences 499-507 and 523-532 of 16S rRNA responsible for binding of ribosomal "core" protein S4 starting the assembly of 30S ribosome subunit. With the simultaneous use of the last two oligonucleotides, the growth of mollicutes in SM IMV-72 medium rich in exogenous sources of nucleosides was suppressed for over 90%. It is supposed that under conditions where mollicutes have no free access to starting materials for their own synthesis of nucleic acid these nucleotides could suppress microorganisms completely. Antisignature oligonucleotides are considered as superspecific agents not leading to the development of resistance of mollicutes and believed to be the main future remedy against diseased caused by microorganisms lacking the system of nucleoside synthesis.

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It is shown that the concentration of "antisignature" phosphorothioate analogs of oligodeoxynucleotides, complementary to the region of 165 rRNA Acholeplasma laidlawii PG-8 and Mycoplasma fermentans PG-18 responsible tor binding with ribosomal protein S4 being 0.5--1 microM synthesis of proteins in vivo decreases to 70%. A model of mechanisms is suggested to block oligonucleotides of the process of in vivo translation in mollicutes by "antisignature" phosphorothioate analogs. The advantages of the use of antisense oligonucleotides complementary to functionally significant plots of 16S rRNA to inhibit the in vivo translation are discussed in comparison with oligonucleotides, 5-nontranslated regions of mRNA serving a target for them.

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Two serine hydrolases have been separated from the proteolytic complex of the venom of Levantine viper, Vipera lebetina turanica. The enzyme with mol. weight of 50,000 +/- 5,000, pH-optimum of 8.5 and isoelectric point in the range of 5.6--6.6 had proteolytic activity against casein and hydrolyzed benzoyl-arginine p-nitroanilide. The other enzyme with mol. weight of 37,000 +/- 2,000, pH optimum of 9 and isoelectric point in the range of 4.1--4.5 had no effect on benzoylarginine p-nitroanilide, casein or hemoglobin, but possessed a bradykinin-releasing activity. Both enzymes were stereoselective against L-arginine, hydrolyzing tosyl-L-arginine methyl ester without having any effect on D-arginine ester. The interaction of the enzymes with a number of N(alpha)-arylsulfonylarginine methyl esters has been studied. The influence of the substitute X in the arylsulfonyl part of the substrates upon their hydrolysis by the bradykinin-releasing enzyme has been described by the Hammett equation of rho omicron = 1.14 +/- 0.33 (r = 0.974).

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The N(alpha)-arylsulfonyl-L-arginine ethyl- and propyl esters were synthesized and the kinetics of their hydrolysis by thrombin was studied. The values of kcat and Km were shown to depend on the structure of the leaving group and to decrease in the line: OCH3 greater than OC2H5 greater than OC3H7. Using methanol as an additional nucleophile, the kinetic parameters - k2, k3 and Ks - were measured for both thrombin- and trypsin-catalysed reactions. A similarity of two enzymes at the stage of Michaelis complex formation was revealed: the Ks values for both enzymes were practically identical (18.10(-5)M). The differences between thrombin and trypsin were observed at the stages of chemical conversion of substrates and were especially well-pronounced at the stage of acylation. It was shown that the k2 values for thrombin were lower than that for trypsin and the k2/k3 ratio of TAME hydrolysis by trypsin was equal to 21, while that for thrombin was 4.5. This finding is indicative of an essential role of the acylation step in thrombin-catalysed hydrolysis of the esters under study.

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For comparative studies on the esterase activities of thrombin and trypsin N(alpha)-arylsulfonyl-L-arginine methyl esters were synthetised containing in aromatic ring substituents of different polar nature, size and hydrophobicity. The kinetics of their hydrolysis by thrombin and trypsin were measured. Values of Km and kcat in steady-state conditions were determined. It was shown, that thrombin-catalysed hydrolysis was more sensitive than that of trypsin to the nature of substituents of arylsulfonyl group and determined by their polar and steric effects. A line correlation between specificity constants (kcat/Km) and sigma and Es of substituents were demonstrated. The difference in reactivity of compounds under investigation is suggested to depend on alterations of stability of hydrogen bond between arylsulfonylamide nitrogen atom of substrate and the active center of the enzyme due to changes in the acidity of the arylsulfonylamide group affected by substituent of the benzene ring.

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Trypsin- and thrombin-catalysed hydrolysis of the Nalpha-aryl-sulfonyl-L-arginine methyl esters was studied. Michaelis constants and kcat were determined. The hydrolysis of the substrates by trypsin appears to depend on the polar nature of substitutes. The thrombin- catalysed reaction was believed to be sensitive to steric effects of the introducted groups.

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