[EFFECT OF MYCOPLASMA INFECTION TO FATTY ACID COMPOSITION OF CALLUS CULTURE SUGAR BEET].

It was studied the effect of Acholeplasma laidlawii var. granulum str. 118 to fatty acid composition of sugar beet calluses. It was established that acting of acholeplasma results to changes in the quantitative content of the individual fatty acids and in the qualitative composition of fatty acids in the lipids of calluses. The changing of the fatty acid composition of calluses lipids of sugar beet infected by A. laidlawii vargranulum str. 118 is observed as nonspecific response to biotic stress.

[Activity of phenylalanine-ammonia-lyase in callus cultures of sugar beat infected by Acholeplasma].

The effect of Acholeplasma laidlawii var. granulum 118 on activity of phenylalanine-ammonia-lyase (PAL) in callus cultures of sugar beat was researched. The optimal conditions of enzyme reaction were: using the L-phenilalanine as a substrate, pH 8.4-8.8, the temperature optimum 38-40 degrees C. It was established that at the infecting of sugar beat callus culture by phytopathogenic mollicute the PAL activity was temporarily increased and reached its maximum after 2 h of infecting. Then it gradually decreased and in 24 h reached its initial level. An increase of PAL activity of plant is considered as protective reaction in response to the action of pathogen.

[Effect of Acholeplasma laidlawii var. granulum on lectin activity of sugar beet calluses].

The dynamics of lectin activity of sugar beet calluses infected by mollicute Acholeplasma laidlawii var granulum str. 118 was studied. Activity of acid-soluble lectins of sugar beet cell cultures increased 4.5 times during some first hours after infecting by acholeplasma. At early stages of infecting by mollicute qualitative changes were also revealed in the protein spectra of acid-soluble lectins of sugar beet calluses.

[Elaboration of the in vitro model system to study the interaction of phytopathogenic mollicutes with plant cells].

The model system based on the sugar beet calluses infected by mycoplasms (mollicutes) was elaborated, and changes in the callus cells morphology under the effect of these microorganisms were also studied. The calluses of sugar beet 3K51 cultivated on the Gamborg medium were infected by phytopathogenic mollicute Acholaplasma laidlawii var. granulum str.118. Under the effect of mollicute infection one could observe changes in the cell morphology of sugar beet calluses: the plant cells were transformed from round to lengthened, the intensity of polyploids forming was increased, their grouping and their total destruction were observed. Data of electron microscopy confirm the presence of the mollicute in the sugar beet calluses: acholeplasma cells were localized between and within undifferentiated plant cells.

[Antineoplastic properties of extracellular fructoso-1,6-bisphosphatase of Bacillus subtilis 668 and preparation isolated from cultural liquids of Bacillus subtilis 7025].

It was shown for the first time that extracellular FBPase of B. subtilis 668 like the preparation obtained from culture liquid of B. subtilis B 7025 displays citotoxicity activity in respect of tumor cells of sarcoma 37 in vitro. It is shown that the preparations remove TA antigens from the surface of the tumor cell. It is supposed that the mechanisms of citotoxic effect of extracellular FBFase of B. subtilis 668 and preparation from the culture liquid of B. subtilis B 7025 in vitro on cells of sarcoma 37 is probably realized through the apoptosis.

[Comparative investigation of fructosobisphosphatases of Bacillus subtilis and pale-green dwarfism pathogens of cereals in the reaction of double diffusion in gel according to Ouchterlony].

Serological properties of fructosobisphosphatases (FBPases) of Bacillus subtilis 668 and PGD agent of cereals--the mollicute Acholplasma laidlawii var. granulum st. 118 (Alg 118) were studied in a comparative aspect with the help of the reaction of double diffusion in gel according to Ouchterlony. It was established for each of microorganisms that their extracellular and intracellular enzymes are similar in serologic respect, and each of them is composed of two antigens, one of them being identical in the both microorganisms, while the other displays only partial identity, since it reacts with antibodies in heterological systems with formation of a precipitation line looking as a "spur". That indicates to the fact that antisera to those enzymes contain antibodies both to general determinants of antigens which are compared (FBPases here), and to the determinant absent in one of them. Basing on the investigation results it is concluded that FBPase of B. subtilis is rather similar than identical, in serological aspect, to the enzyme Alg 118 of the same name.

Antisignature oligonucleotides and their analogs as inhibitors of mollicutes-cofactors of HIV.

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.

[The binding and absorption by mollicute cells of antisignature analogs of oligodeoxyribonucleotides]. / Sviazyvanie i pogloshchenie kletkami mollikutov antisignaturnykh analogov oligodezoksiribonukleotidov.

Processes of absorption and uptake of phosphorothioate, phosphorodithioate and methylphosphonate analogs of oligodeoxynucleotides which are complementary to "signature" 16S rRNA sequences by cells of Acholeplasma laidlawii PG-8. Mycoplasma fermentans PG-18 and Mycoplasma pneumoniae FH have been investigated. Distinctions in efficiency of absorption by given cells of phosphorothioate and methylphosphonate analogs of oligonucleotides are found out. So, the level of thioates sorption by the cells of A. laidlawii PG-8 exceeded a share of methylphosphonates binding with these cells 5 times as high. A fact of significant accumulation of thio- and dithioate analogs of oligonucleotides by the mollicute cells is established. The intracellular concentration of these compounds in all investigated microorganisms exceeded extracellular one 10(4)-10(5) time. On the basis of obtained experimental data the assumption is made on existence in mollicute cells of two various mechanisms of uptake of analogs of oligonucleotides bearing a negative charge in sugar-phosphate backbone and nonionic oligonucleotide analogs.

[The validation of the possible use of monosugars and 6-azacytidine for the elimination of Mollicutes associated with HIV/AIDS from the human urogenital tract]. / Obhruntuvannia mozhlyvosti zastosuvannia monotsukriv i 6-azatsytydynu dlia eliminatsiï z urohenital'noho traktu liudyny molikutiv, iaki asotsiiuiut' z VIL/SNIDom.

A search for the methods new in principle which should block and eliminate AIDS-associated mycoplasmas was carried out. This work was conducted in two ways: 1) inhibition of vital activity of Mycoplasma fermentans PG-18 and Acholeplasma laidlawii PG-8 by 6-azacytidine; 2) establishment of carbohydrate composition of receptors for these mycoplasmas aimed at the competitive elimination of these microorganisms from urogenital tract of a man using carbohydrates. It is established that a 50%-inhibiting concentration of 6-azacytidine was 23.4 micrograms/ml for M. fermentans PG-18 and 62.5 micrograms/ml for A. laidlawii PG-8. alpha-D-glucose and N-acetylneuramine acid are two terminal carbohydrates that can serve as receptors for M. fermentans on human mucous membranes while D-mannose and N-acetyl-D-glucosamine for A. laidlawii PG-8. alpha-D-glucose in concentration 75 mM and N-acetylneuramine acid in concentration 150 mM competitively inhibit reception of M. fermentans on mucosae, while D-mannose in concentration 150 mM and N-acetyl-D-glucosamine in concentration 75 mM are antireceptor substances for A. laidlawii.

[The sorption of antisense oligodeoxyribonucleotides by Mycoplasma cells]. / Issledovanie sorbtsii antismyslovykh oligodeoksiribonukleotidov kletkami mikoplazm.

The paper deals with kinetics of binding of antisense oligodesoxyribonucleotides, complementary to certain sequences 16 S RNA of mollicutes, by the cells of three representatives of class Mollicutes: Acholeplasma laidlawii PG-8. Mycoplasma pneumoniae FH and M. fermentans PG-18. It is shown that binding of antisense oligonucleotides by the mollicute cells depends on temperature and age of cultures. The highest level of sorption of labelled antisense oligodesoxyribonucleotides by the cells of mycoplasmas corresponded to the phase of logarithmic growth of each of the studied mollicute strains. The lengthening of nucleotide chain from 5 to 15 nucleotide bases did not result in the decrease of sorption of the studied oligodesoxyribonucleotides by the mollicute cells.

[The ability of the oligodeoxyribonucleotides complementary to the 3'-terminal segment of 16s-rRNA in Mollicutes to suppress translation in their ribosomes in an in-vitro system]. / Sposobnost' oligodezoksiribonukleotidov, komplementarnykh 3'-kontsevomu uchastku 16S-rRNA mollikutov, podavliat' transliatsiiu na ikh ribosomakh v sisteme in vitro]

Effect of oligodesoxyribonucleotide, complementary to 3'-end sequence (3'-UCUUUCCUCCAC) of 16S-rRNA of mollicutes, and its phenasine derivatives on the process of translation of mollicute has been studied in the system of in vitro translation, created on the basis of ribosomes of Acholeplasma laidlawii var. granulum str. 118 and germ extracts of the wheat and optimized in respect of the temperature (23 degrees C), translation time (70 min), concentration of potassium and magnesium ions (150 and 5 mM, respectively). It is shown that acholeplasma ribosomes are most efficiently inhibited (60%) under their interaction with oligonucleotide containing one phenasine insert on the 3'-end, nonmodified oligonucleotide exerted a bit less inhibiting effect (58%). Oligonucleotide containing intercalating inserts in 3'- and 5'-positions manifested the least inhibiting effect (35%). It is noted that the efficiency translation inhibition by synthetic oligonucleotides is conditioned by nuclease activity of the system and by the length of the section of active binding of oligonucleotide with the sequence target on rRNA. It is supposed that oligonucleotides complementary to certain unique rRNA sequences can become promising highly specific drugs for prophylaxis and treatment of mycoplasmoses.

[Oligodeoxyribonucleotides complementary to sections of the mollicute ribosomal operon as transcription inhibitors in vitro]. / Oligodezoksiribonukleotidy, komplementarnye uchastkam ribosomal'nogo operona mollikutov, kak ingibitory transkriptsii in vitro.

Antisense oligodeoxyribonucleotides have been studied for their effect on the transcription in vitro in mollicutes. A synthetic fragment of DNA [symbol: see text] complementary to that part of DNA which codes the 1510-1521 area of the 3'-terminal sequence 16S-pRNA of all mollicutes was used in the study as well as its modifications by imidasophenasine derivatives: [symbol: see text], [symbol: see text] [symbol: see text]. Maximal inhibition of the mollicute transcription in vitro was observed with 100 nM oligonucleotide concentration. Lower or higher concentrations were less effective. Transcription initiated by RNA-polymerase of M. fermentans PG-18 (a mollicute strain referring to AIDS disease) proved to be the most sensitive to the effect of modified oligonucleotide: it was inhibited by 75-80%. It is concluded that modified oligonucleotides exert a dual effect on transcription: firstly, they participate in nonspecific interaction with RNA-polymerase which induces insignificant inhibition of transcription and, secondly, they complementary interact with homologous sections of one-stranded DNA-matrix and block the RNA synthesis. Binding of modified oligonucleotides with DNA is rather strong.

[The inhibitory action of 6-azacytidine on Mollicutes and its proposed mechanism]. / Ingibitornoe deistvie 6-azatsitidina na mollikuty i ego predpolagaemyi mekhanizm.

6-Azacytidine (6-AC) is shown to have an inhibitory effect on the Mollicutes of the different systematic position. The growth of type strains of Mollicutes (Acholeplasma laidlawii PG-8, Mycoplasma pneumoniae FH and M. fermentans PG-18) completely ceased in the nutrient medium at concentration of the above substance in it within the range of 125-250 micrograms/ml. 50% inhibiting concentration of 6-AC equaled: for M. fermentans PG-8: 23.43 micrograms/ml; M. pneumoniae FN: 46.8 micrograms/ml; Acholeplasma laidlawii PG-8: 62.5 micrograms/ml. 6-AC concentration 5 micrograms/ml decreased the process of DNA-dependent DNA synthesis in the in vitro system more than by 60%. 6-AC exerted less effect on the DNA-dependent RNA synthesis in the in vitro system: at different concentrations of 6-AC (up to 400 micrograms/ml) RNA synthesis decreased only by 20%. Translation on ribosomes of Mollicutes in the in vitro system completely ceased at 6-AC concentration 100 micrograms/ml. The results obtained indicate that for 6-AC in cells of Mollicutes and, possibly, for other microorganisms there are two targets: ribosomes and DNA-dependent DNA-polymerase. Total effect of blocking of the translation and replication processes by 6-azacytidine causes death of Mollicutes. Since 6-AC has no harmful effect on the human cells, it can be used as an efficient method for treatment of respiratory and urogenital diseases induced by Mollicutes.

[The isolation of ribosomes from Acholeplasma and the study of their physicochemical characteristics]. / Poluchenie ribosom akholeplazm i issledovanie ikh fiziko-khimicheskikh kharakteristik.

Ribosomes are produced from Acholeplasma laidlawii PG-8 and A. laidlawii var. granulum str. 118. It is proved as possible to use the standard method of differential centrifugation for isolation of mollicute ribosomes. The physico-chemical properties of acholesplasma ribosomes are studied. The protein content for A. laidlawii PG-8 amounted to 39.6%, rRNA content--60.4% and for A. laidlawii var. granulum--39.1 and 60.8%, respectively. The RNA: protein ratio equals 1.5:1, the ratio of optic density indicates at 260 and 280 nm--2, that is peculiar to treated preparations of ribosomes. Sedimentation coefficient of acholeplasma ribosomes is 70S. The produced preparations of acholeplasma ribosomes can be used subsequently for creating the system of translation in vitro to study the biosynthesis processes of mollicute protein.

[The stability of the alkylating derivatives of oligodeoxyribonucleotides containing a cholesterol or phenazine radical added to the 3'-termination during their interaction with Acholeplasma laidlawii PG-8]. / Issledovanie stabil'nosti alkiliruiushchikh proizvodnykh oligodezoksiribonukleotidov, soderzhashchikh prisoedinennyi k 3'-kontsu ostatok kholesterina ili fenaziniia, pri vzaimodeistvii ikh s kletkami Acholeplasma laidlawii PG-8.

Stability of alkylating derivatives of decathymidylates protected on the 3'-terminal by cholesterol and phenazine residues has been studied in the process of their interaction with cells of Acholeplasma laidlawii PG-8. It is shown that the studied reagents are not split by nucleases of A. laidlawii PG-8 for the time necessary for alkylation of mycoplasma biopolymers.

[The interaction of alkylating derivatives of oligodeoxyribonucleotides and their methylphosphonate analogs with Mycoplasma cells]. / Vzaimodeistvie alkiliruiushchikh proizvodnykh oligodezoksiribonukleotidov i ikh metilfosfonatnykh analogov s kletkami mikoplazm.

Alkylating derivatives of decathymidylates and methylphosphonate analogs of oligodeoxyribonucleotides (MPAO) were studied for their interaction with cells of Acholeplasma laidlawii PG-8, Mycoplasma capricolum California Kid, M. pneumoniae FH and phytopathogenic strain (St. 118). It is shown that MPAO of octa- and hexadecathymidylates as well as decathymidylates 3'-terminal modified by phenazine and cholesterol groupings are sorbed by mycoplasma cells and can penetrate inside the cells. Efficiency of binding of alkylating derivatives and MPAO with mycoplasma cells depends on interaction time of reagents, their concentration in the reaction mixture and temperature.

[The DNA nucleotide composition of bacteria oxidizing diethylene glycols]. / Nukleotidnyi sostav DNK bakterii, okisliaiushchikh diétilenglikol'.

Nucleotide composition of DNA has been analyzed in bacteria composing the diethylene glycol-oxidizing association. It is shown that the microorganisms under study are representatives of genera Alkaligenes and Achromobacter.

[The thermodynamic parameters of the conformational transitions in Mycoplasma DNA]. / Termodinamicheskie parametry konformatsionnykh perekhodov DNK mikoplazm.

DNA preparations have been isolated from 10 strains of phytopathogenic mycoplasms and collection culture Achole plasma laidlawii PG-8. Thermodynamic parameters of denaturation changes in the secondary structure (transconformation) of DNA of these mycoplasms have been determined. It is shown that denaturation temperature is 82.3-83.1 degree C and enthalpy of conformational DNA transitions calculated per 1 g of dry substance is 56.2-61.9 J/g. Changes in the enthalpy (delta H) and entropy (delta S) calculated per 1 mol of nucleotide pairs varied in the range of 35.6-39.0 J/m.p. and 995-109.6 J degree m.p. respectively. No linear dependence of transconformational thermal adsorption on the temperature of DNA denaturation of the studied strains of mycoplasms are revealed, which is probably connected with structural peculiarities of DNA of these microorganisms.