[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.

[Amino acid assimilation by representatives of the Acholeplasma genus].

Amino acid assimilation by different representatives of Acholeplasma genus has been investigated. It was shown that all 7 investigated typical strains Acholeplasma laidlawii PG-8, A. granularum BTS-39, A. modicum PG-49, A. oculi 19-L, A. morum 72-043, A. axanthum S-743, A. hippikon C-1 and 37 phytopathogenic strains isolated from the affected plants were able to assimilate asparagine, glutamine, threonine, histidine and proline. A majority of acholeplasmas were able to metabolize phenylalanine, methionine, glutamate and lysine, rarely isoleucine. Each of the investigated species of acholeplasmas had individual spectrum of assimilated amino acids that can be used as an additional systematic characteristic of mollicutes.

[Molecular weight and subunit composition of extracellular fructosobisphosphatase of Acholeplasma laidlawii var. granulum strain 118].

Molecular weight of extracellular fructosobisphosphatase of Acholeplasma laidlawii var. granulum strain 118--an agent of pale-green dwarf of cereals has been determined. This enzyme is the basic factor of pathogenicity of this organism, and, maybe, of all phytoplasmas, owing to realization of the enzyme noncontrolled function in the plant organism, its habit acquires the disease symptoms characteristic of "yellows". It has been established that in the native condition the extracellular fructosobisphosphatase of Acholeplasma laidlawii var. granulum st. 118 has molecular weight 230 000 +/- 5 000 Da, and in denaturating ones--56 600 Da, i.e., the enzyme in the native condition consists of four equal subunits. The subunit composition is peculiar to fructosobisphosphatase of other microorganisms already described in literature; fructosobisphosphatase of the mollicute as to its parameters both in native and denaturated conditions occupies the intermediate place among them.

[Effect of separate effectors on activity of extracellular fructosobisphosphatase of Acholeplasma laidlawii var. Granulum, strain 118].

The influence of 16 substances-effectors on the extracellular mollicute fructosobisphosphatase (FBPhase) was studied for the first time. These effectors are used, as a rule, when studying properties of this enzyme biopreparations newly isolated from the cells of animals, plants and cells of microorganisms. It was established that optimum pH for FBPhase of mollicutes is whithin 7.3-7.5 and on the basis of this index it is attributed to the group of the "neutral" of these enzymes. Cations of K, Mn, Mg, NH4, Tl and phosphoenolpyruvate (PEP) increase its activity. Cations of Li and adenosin 5'-monophosphate (AMP) proved to be the inhibitors of mollicute FBPhase activity. Chelators (EDTA, citrate, imidasol pyruvate, L-histidine) activated it inconsiderably (by 10-20%). Cations of Zn, in contrast to those of other tested metals, in low concentrations (0.1 - 0.2 microM) inhibited FBPhase activity, but when increasing their concentration (6 microM and above) activated the enzyme even better than it was observed for Mn and Mg cations, the necessary components of the reacting mixtures. Thus, when determining the components of the reacting mixtures with the purpose to study the properties of mollicute FBPhase and to regulate its activity in the in vitro systems under pH values optimal for the enzyme, the monovalent (NH4, K, Tl) and bivalent (Mg, Mn, Zn) cations may be introduced in their compositions as activators, AMP and Li cations should be used as inhibitors. Other substances which were studied when making their work proved to be inessential effectors is respect of mollicute FBPhase.

[Effect of silver nitrate, adenosine-5'-monophosphate and phosphoenolpyruvate on activity of extracellular fructose bisphosphatase of Acholeplasma laidlawii var. granulum strain 118].

The reactions of glycolysis or gluconeogenesis proceed in good coordination in the cells of microorganisms, and each stage of these processes is distinctly regulated. Under such conditions fructose-bisphosphatase (FBPase) activity (the enzyme level being constant in the cells of microorganisms) is inhibited by adenosine-5'-monophosphate (AMP) and is activated by phosphoenolpyruvate (PEP) depending on the kind of the source of carbon (glycolytic or glyconeogenic) used for microorganism growth. It is evident that the corresponding regulation of FBPase should be absent in the extracellular environment where one cannot observe a distinct coordination of functioning of the enzyme systems. The investigation results prove that both AMP and PEP, under their individual testing in concentrations up to 20 microM did not practically affect activity of extracellular FBPase, and at higher concentrations they sharply decreased its activity (200 microM AMP by 70%, and PEP - by 75%). Under joint use of PEP and AMP (in concentration 200 microM and 500 microM) one could observe mutual neutralization of the effect of these substances on FBPase; as a result, its activity decreased only by 15% under AMP concentration of 500 microM, and by 25% at AMP concentration of 200 microM, that is in complete agreement with the data of individual testing of the above substances. PEP in high concentrations has displayed itself as a more active repressor of FBPase activity than AMP. AgNO3 in concentrations to 20 microM has manifested itself as a moderate stimulator of FBPase activity and even in the concentration of 200 microM it decreased the enzyme activity by 50% only. The data obtained are rather different than those described in literature for cellular FBPases of microorganisms. It is known that AMP is a powerful inhibitor of its FBPases activity (Ki = 5 microM) while PEP activates it (Ka = 20 microM).

[The lectins of Mollicutes]. / Lektyny molikutiv.

Lectine-carbohydrate interactions take the fundamental part in the intercellular relations as well as in microbes pathogenicity. Pathogenic Mollicutes are characterized by the firm adhesion to the cells of the affected organs of people, animals, insects and plants. Proteins which are localized on the external side of the mollicute membrane and interact with carbohydrate residues on the surface of mucous membranes of the damaged organs and vice versa take part in the adhesion. High degree of specificity of protein-carbohydrate interactions determines the pathogenic specializations to the cells of one or another organ of the host. Since proteins which take part in the mollicutes adhesion are rich in proline and hydrophobic fields it is not excluded that the adhesion processes are combined with hydrophobic interrelations between the cells of the pathogen and host. Substances from the cells of Mollicutes which completely correspond to the definition "lectin", i.e., are in a pure form, the carbohydrate-binding proteins specific to certain carbohydrate residues are not still isolated. Thus the mollicute lectines should be called lectine-like substances rather than lectines. Mollicutes form a lot of such substances and they may be separated into the extracellular (soluble) substances which are found outside the cell of the nutritious medium; intermediate (half-soluble) substances detected in the nutritious medium and in the state integrate into the microorganism membrane, and membrane-related (insoluble) substances which occur only in the state rigidly adhered to the membrane. Carbohydrate composition of lectine-like substances in different mollicutes is different which is the reflection of heterogenicity of the Mollicutes class representatives.

[Features of amino acid assimilation by representatives of the Mycoplasma genus]. / Osoblyvosti zasvoiennia aminokislot predstavnykamy rodu Mycoplasma.

Amino acid assimilation by different representatives of Mycoplasma genus has been investigated. All typical strains, involved in this research--Mycoplasma pneumoniae, M. capricolum, M. hominis, M. mycoides subsp. capri, M. fermentans, M. salivarium were able to assimilate asparagine, glutamine, threonine, histidine and tryptophan. Most of the investigated mycoplasmas were able to assimilate proline, phenylalanine, methionine, glutamate, lysine, serine, tyrosine, glycine, valine, isoleucine and alanine; assimilation of leucine and cysteine was observed rarely. Each of the investigated species of mycoplasmas are characterized by a specific spectrum of assimilated amino acids that can be used as additional characteristic for systematics of mollicutes.

[Soluble and semisoluble lectins from mollicutes and their possible functions]. / Rozchynni ta napivrozchynni lektyny molikutiv i ïkh mozhlyvi funktsiï.

When studying mollicute lectins it was established that Acholeplasma laidlawii PG-8 synthesizes two half-soluble lectins one of which is specific to N-acetyl-D-glucosamine, and the other--to D-glucosamine.HCl; phytopathogenic strain A. laidlawii var. granulum 118 produced 4 lectins one of which is soluble and specific in respect to fructose-1.6-diphosphate, the rest three lectins are half-soluble and specific to one of the sugars--D-galactosamine.HCl, rafinose and D-glucosamine.HCl. In Mycoplasma pneumoniae FH all the four lectins found in the culture liquid have been classified as half-soluble, specific to one of carbohydrates--D-galactosamine.HCl, talose, N-acetyl-neuramine acid and D-glucose; M. capricolum Cal. Kid. synthesizes four lectins; two of them being defined as soluble (one of the lectins is, respectively, specific to talose and D-glucosamine.HCl, two others, as half-soluble, specific to one of sugars--rafinose or D-glucose. The results obtained permit a conclusion to be made that the half-soluble lectins of mollicutes, on the one hand, are the factors of adhesion on the corresponding organs of macroorganism and, on the other hand take part in the transport of substances from without into the microorganism cell. Soluble lectins determine pathogenicity of mollicutes and form with half-soluble lectins a single chain to providing the mycoplasma cells with nutrients and to protect them from the action of the macroorganism immune system.

[Method for obtaining the basic factor of pathogenicity of Phytoplasma--extracellular fructose-1,6-bisphosphatase (on the model of the agent of pale-green dwarfness of cereals in culture)]. / Spoib oderzhannia pozaklitynnoï fruktozo-1,6-bisfosfatazy--osnovnoho faktora patohennosti fitoplazm (na modeli zbudnyka blido-zelenoï karlykovosti zernobykh kul'tur).

A preparation with fructose-1,6-bisphosphatase (FBPase) activity has been isolated as a result of the five-stage treatment of the culture liquid obtained after growing the agent of pale-green dwarfness of cereals on the medium CM IMB-72. After this enzyme treatment by means of hydrophobic chromatography on the column from Toyopearl HW-60 (the enzyme was obtained from the column with decrease of ammonium sulphate (AS) concentration in the eluating buffer to 0.8 M), the preparation deprivation of AS on the column with Sephadex G-10 and substrate-dependent chromatography on the column with CM-sepharose the extracellular 176-fold purified FBPase. Acholeplasma laidlawii var. granulum strain 118 was obtained--the main pathogenicity factor for the agent of cereals yellow.

[Carbohydrate receptors for Mycoplasma fermentans adhesion on human epithelial tissues]. / Vuhlevodni retseptory dlia adheziï Mycoplasma fermentans na epitelial'nykh tkanynakh liudyny.

It has been stated that two terminal carbohydrates from polysaccharide complexes which are on the surface of human epithelial tissues, namely, alpha-D-glucose and N-acetylneuramino acid bound with subterminal galactose via alpha 2-->3-bond (NeuAc alpha 2-->3 Gal), may serve receptors for Mycoplasma fermentans adhesion on human epithelial cells. M. fermentans shows high selectivity to these receptors, though very low affinity. The latter, probably, explains why this mycoplasma is able to infect only the limited number of peoples. In the authors' opinion people with the lower content of glucose in urine, as well as those who suffer from diseases associated with hypothalamo-hypophyseal insufficiency are subjected to infection with M. fermentans. People with normal (3.33-5.55 mM) and elevated alpha-D-glucose content in blood and in urine are not susceptible to this mycoplasma. Results of the research carried have shown that alpha-D-glucose solutions of definite concentration may be used to eliminate M. fermentans from the urogenital tract of people who have it. The ability of M. fermentans to discriminate terminal structure of NeuAc alpha 2-->3 Gal provides it with the possibility to adhere human immunodeficiency virus virions on its cells as glycoprotein (gp120) of that virus has among its own oligosaccharides certain glycopolymers of the similar terminal structure. Then M. fermentans transports the virions directly to target cells for this virus. The target cells express receptor CD4 glycolized by oligosaccharides of the mentioned terminal structure. It provides adhesion of the mycoplasma on the receptor.

[The phenotypic traits of Mollicutes as their possible phylogenetic markers]. / Deiaki fenotypovi oznaky molikutiv iak ïkh mozhlyvi filohenetychni markery.

Interaction of cells of mollicutes Acholeplasma laidlawii PG 8, A. laidlawii var. granulum 18, Mycoplasma hominis PG 21, M. pneumoniae FH, M. fermentans PG 118 and their extracellular products with different carbohydrates, plant lectins of different carbohydrate specificity with glycocalix carbohydrates of the same microorganisms has been studied. Basing on this study and data from literature a conclusion is made that such phenotypical characteristics as the ability to form extracellular fructose-1.6-diphosphate specific lectin and N-acetylneuraminic acid as the end sugar in the composition of carbohydrates of mollicute glycocalyx can serve a phylogenetic marker. These markers indicate the possible origin of mollicutes from bacteria of the group Bacillus-Lactobacillus-Streptococcus as a result of degenerative evolution and are their rather stable characteristics. Such marker as extracellular lectin specific to fructoso-1.6-diphosphate which is formed by phytopathogenic mollicute A. laidlawii var. granulum, 118 evidences that in spite of genetic affinity of this "yellow" agent of cereals with A. laidlawii, it does not descend from the last ancestor directly, but they probably have some general ancestor. We do not know yet this ancestor which is a link in the evolution chain of acholeplasmas in the process of their origin from the mentioned group of bacteria. It is supposed that these markers together with such known phylogenetic markers as lactate dehydrogenase which is activated by fructoso-1.6-diphosphate, and aldolases and glycolipids with specific properties can additionally evidence for the origin of mollicutes and their affinity to certain groups of microorganisms.

[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 inhibition of Mycoplasma pneumoniae adhesion in a fetuin test system by synthetic analogs and polymeric forms of neuraminic acid]. / Ingibirovanie adgezii Mycoplasma pneumoniae v fetuinovoi test-sisteme sinteticheskimi analogami i polimernymi formami neiraminovoi kisloty.

Eight glycosides and structural analogues of neuraminic acid as well as eight polymeric forms of N-acetyl neuraminic acid have been studied for their inhibitory effect on adhesion of Mycoplasma pneumoniae. Maximum inhibiting effect among low-molecular compounds was manifested by 2-->3 sialyllactose which, being used in concentrations 5.0 and 10.0 micrograms/ml, inhibited adhesion of mycoplasmas by 76 and 87%, respectively. These indices for other derivatives in the above mentioned concentrations were as follows (%): 2-->6 sialyllactose, 31 and 74%; alpha-Me-glycoside NeuAc, 75 and 85%; alpha-Bn-glycoside-N-trifluoruracetyl NeuAc, 30 and 63%; alpha-Bn-glycoside NeuAc, 32 and 59%; alpha-Bn-glycoside-4-epi-NeuAc, 20 and 27%; beta-Bn-glycoside NeuAc, 2-4%; beta-me-glycoside NeuAc, 4-5%. The maximum inhibiting effect (50% inhibition at concentration 2.5 mumol) among polymeric forms was exerted by the conjugate alpha-benzeneglycoside with polyacrylic acid containing 12 mol% of NeuAc. Conjugates with 8, 16 and 20 mol% of NeuAc possessed a bit less activity. The 50% concentration for them was 5.3, 3.1 and 8.3 mumol, respectively. Polymeric forms on the basis of polyacrylamide proved less active.

[Mono- and polyvalent synthetic sialosides as inhibitors of Mycoplasma pneumoniae adhesiveness]. / Mono- i polivalentnye sinteticheskie sialozidy kak ingibitory adgezivnosti Mycoplasma pneumoniae.

Nine mono- and polyvalent sialosides as substances inhibiting adhesive properties of mycoplasma Mycoplasma pneumoniae--the agent of human atypical pneumonia have been studied in the reaction of hemagglutination (RHA) using solid-phase variant of ELISA-test ("sandwich"--variant), which is based on the competition for specific binding in RHA between the studied syalosides and sialylglycoproteins of fetuin conjugate with horseradish peroxidase. Of seven polymers--P alpha.12.ONa(N1), P alpha.12.EA(N2), P alpha.12.NH2(N3), P alpha.12.AES.10.ONa(N4), P alpha.12.Hg.20.ONa(N6), P20.SLea.NH2(N7), as well as monomer alpha-Bn Neu5Ac and fucoidan the polymers 5, 2, 4 and 7 in concentrations as to the content of sialic acid 1.0; 1.3; 1.35 and 10.0 M, respectively, most efficiently (up to 98%) inhibited the adhesiveness of M. pneumoniae. Polymeric sialosides 3, 6 and 1 proved less active and, the concentration of sialic acid in the composition of their molecules being 10.0 microM, inhibited adhesiveness of M. pneumoniae by approximately 77, 75 and 62.5%, respectively. Antiadhesive activity of fucoidan and monomer proved too low under concentration of these substances as to the content of sialic acid in them 25 microM: they decreased the ability to adhesion in M. pneumoniae by 33 and 56%, respectively. This proved that polyvalent sialosides 2, 4, 5 and 7 are promising for creation of drugs for treatment and prophylaxis of human pneumonias of mycoplasmal etiology on principally new basis with regard for the properties of the disease agent.

[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.