[The Effect of Cadmium on the Efficiency of Development of Legume-Rhizobium Symbiosis].

Screening of nodule bacteria (rhizobia) forming symbiotic relationships with legumes has been performed in order to isolate strains resistant to cadmium ions in a wide range of concentrations (6-132 mg/kg). The effect ofcadmium salts (6, 12, 24 mg/kg) on the legume-rhizobium symbiosis ofthe pea Pisum sativum L. with Rhizobium leguminosarum and of the fodder galega Galega orientalis Lam. with Rhizobium galegae has been studied under experimental laboratory conditions. No statistically significant differences have been revealed in the growth and biomass of plants with regard to the control in the range of concentrations given above. However, it was found that cadmium inhibited nodulation in P. sativum and stimulated it in G. orientalis.

[THE MOLECULAR GENETIC CHARACTERISTIC OF SPECIES CONTENT OF SALIVA AND GINGIVAL RECESS UNDER PERIODONTITIS].

The examination was carried out of samplings of 110 patients with periodontitis (observation group) and 60 patients without pathology of periodont (comparison group). The polymerase chain reaction was used to analyze samples of saliva and contents of periodontal recesses for detecting species-specific DNA fragments of Porphymmonas gigngivalis, Streptococcus macacae, S. mutans, S. oralis, S. salivarius, S. sangis, S. sobrinus, Treponema denticola. In patients with periodontitis S. mutans, S. oralis S. sobrinus were reliably more often detected in the content of periodontal recesses and S. mutans, S. sobrinus i in saliva. In the observation group the rate of detection of association S. mutans--S. oralis--S. sangis--S. sobrinus was significantly exceeded (up to 15.6%, X2 = 9.1, p = 0.004). In ten days of effective treatment of periodontitis reliable decreastng of rate of detection of S. wasoralis, S. sobrinus was observed in contents of periodontal recesses but not in of saliva. The detection of S.sobrinus using technique of polymerase chain reaction in contents of periodontal recesses and/or saliva of patients with periodontitis has a diagnostic value. The detection of S.sobrinus in contents of periodontal recesses is significant both in monoculture and in association S. mutans--S. oralis--S. sangis--S.sobrinus. The absence of S. sobrinus in contents of periodontal recesses testifies effectiveness of treatment of main disease (periodontitis).

[The comparative evaluation of informativity of methods of etiologic diagnostics of community-acquired pneumonia].

The article deals with the results of selection and testing of oligonucleotide primers to gene 16S pRN4 of a number of agents of community'-acquired pneumonia (Streptococcus pneumoniae. Haemorhilus influenzae. Moraxella catarrhalis, Klebsiella pneumonia. Pseudomonas aeruginosa. StaphyIococcus aureus) for their highly specified detection in clinical material on application of polymerase chain reaction. The comparison with bacteriological method (golden standard) is used to demonstrate the high sensibility, specificity and diagnostic effectiveness of product enabling to apply it in practice to diagnose communitly-acquired pneumonia regardless of duration, severity and phase of disease and to develop corresponding diagnostic systems.

[Development and use of test-systems for molecular-genetic detection of out-of-hospital pneumonia agents].

AIM: Search of new targets and improvements of molecular-genetic detection methods of species specific DNA fragments of out-of-hospital pneumonia (OP) infection agents in sputum. MATERIALS AND METHODS: Species specific oligonucleotide primers to Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Branhamella catarrhalis were selected. 103 sputum samples from OP patients and 15 from practically healthy humans were test by using PCR and bacteriological method. RESULTS: Mixed bacterial flora was detected by bacteriologic method in OP in 26 cases (25.2%). 14 patients (13.6%) had B. catarrhalis DNA in sputum, 12 patients (11.6%)--S. pneumoniae, H. influenzae was detected in 11 examined individuals (10.7%), K. pneumoniae (7.9%)-- in 8. In 32 cases (30%) etiology of OP could not be deciphered bacteriologically. B. catarrhalis was detected in 19 (18.5%) of OP patients, S. pneumoniae--in 17 (16.5%), H. influenzae and K. pneumoniae--in 14.5% and 12.6% (15 and 13 patients) respectively by Using the developed PCR primers. In 16.5% cases (17 patients) positive results against several bacteria species simultaneously were obtained by PCR method, in 3.9% (4 patients) P. aeruginosa was detected. CONCLUSION. Use of PCR for detection of out-of-hospital pneumonia agents DNA significantly increases informativity of the study when compared with bacteriological method.

[Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro].

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.

[Effects of natural and hybrid lectins on the legume-rhizobium interactions].

The effects of hybrid lectins--full-sized pea Pisum sativum lectin (PSL) with the carbohydrate-binding region of white melilot Melilotus albus lectin or wild licorice Astragalus glycyphyllos lectin substituted for the corresponding PSL region (PSL/MAL and PSL/AGL, correspondingly)--on the legume-rhizobium symbiosis were studied. The treatment of the Rhizobium leguminosarum bv. viciae in the alfalfa (Medicago sativa) rhizosphere with PSL induced formation of uninfected pseudonodules on its roots, whereas the treatment of the bacteria from Astragalus cicer nodules with PSL/AGL rendered these bacteria able to form infective nodules on alfalfa roots. This ability is associated with expanded and unusual carbohydrate-binding properties (combined specificity for Gal and Glc) of this hybrid protein as compared with the natural legume lectins.

[Genetic markers of pathogenicity of conditionally pathogenic enterobacteria isolated from children with diarrheal diseases].

In the process of examination of 89 children from different age groups with diarrheal disease caused by bacteria from Enterobacteriaceae family 89 microorganisms were isolated including Klebsiella spp. (37 isolates), Citrobacter spp. (9 isolates), Enterobacter spp. (17 isolates), Hafnia alvei (1 isolate), Morganella morganii (11 isolates), Proteus spp. (14 isolates). Presence of genes associated with pathogenicity islands (PAIS): hlyA, hlyB (hemolysin), sfaG (fimbria antigen type S), cnf1 (cytotoxic necrotizing factor 1), estB (heat-stable enterotoxin B)were studied in these cultures by PCR. It was found that 32.6% of examined isolates had fragments of PAIS's genes--hlyA was detected in 9 cases (10.1%), hlyB--in 10 cases (11.2%), sfaG --in 8 cases (9%), cnf1--in 9 cases (10.1%), and estB--in 3 cases (3.4%). Positive correlation between genetic determinants hlyB and cnf1 as well as hlyA and sfaG was found while estB was not associated with other genes. Weak positive correlation between presence of sfaG and resistance to tetracycline and chloramphenicol was detected. Factors coded by revealed determinants of PAIS can play a role in the development of diarrheal syndrome.

[Identification of the causative agent of echinococcosis in the population of the South Urals].

The topicality of the problem associated with echinococcosis granulosus in the South Urals is determined by its wide spread and a considerable economic damage made to this region by this invasion. The study was undertaken to reveal the intraspecific affiliation of Echinococcus granulosus that induces hydatid disease in the population of the South Urals. Samples for studies were taken from the fertile larval cysts obtained during intraoperative intervention in patients with hydatid disease. As morphological criteria for differentiation, the authors examined the proboscis uncuses of protoscolexes. For E. granulosus genomic typing, polymerase chain reaction (PCR) of DNA synthesis was used, as described by Gasser (1998). As a DNA marker, the authors used a fragment of the mitochondrial gene encoding for the first subunit of cytochome-C-oxidase. The DNA fragments obtained by PCR from 9 isolated underwent the direct enzyme dideoxy-sequencing test (Senger, 1977). As a result, the causative agent of echinococcosis granulosis was first identified in the patient of the South Urals. In children and adults, the clinical form of the disease is caused by E. granulosus with the genotype G - common, that of domestic sheep. Comparative analysis of molecular data revealed the presence of genotype G1 variations circulating in the South Urals homologous to the sequences recorded in the GenBank under numbers U50464 and DQ109036.

[Contribution of lectin sugar-binding peptides structure determines specificity of rhizobium-legume symbiosis in Galega orientalis and G. officinalis].

A significant heterogeneity between bacteria Rhizobium galegae bv. officinalis and R. galegae bv. orientalis forming the nitrogen-fixing symbiosis with Galega officinalis and G. orientalis, respectively, and not forming any single cross-inoculation group, was found by means of RAPD and RFEL methods. The high level of sequence similiraty between lectins of these plants indicates at their close relationship. However the sequences of lectin sugar binding peptides (SBP) of G. orientalis (TYCNPGWDPRDR) and G. officinalis (TFYNEEWDLVIKDEH) were highly diverged. Amino acids of SBP which are involved in linkage of Ca2+ and Mn2+ ions responsible for stabilization of spatial structure of carbohydrate-binding "pocket", keep their position in peptide. It suggests that lectins participate in Rhizobium-legume symbiosis and that carbohydrate-binding site plays a key role in this process.

[Phylogeny of Triticum L. and Aegilops L. genuses inferred from a comparative analysis of nucleotide sequences in promoter rDNA regions of individual species]. / Filogeneticheskie vzaimootnosheniia rodov Triticum L. i Aegilops L. i nukleotidnye posledovatel'nosti promotornykh obtastei rDNK ikh otdel'enykh predstavitelei.

The process of accumulation of knowledge on wheat and related wild species during the 20th century is briefly reviewed with special reference to the evidence of the recent years on evolution of polyploid wheats and the role of diploid species. The latter serve as potential donors of the genomes, detection of which is particularly important because of the continuing speciation in the tribe Triticeae and artificial development of synthetic forms. The arguments in favor of the donor role for various diploid wheat species and aegilopses from the section Sitopsis are compared. It is stated that in the formation of the both lines of polyploid wheats turgidum-aestivum and timopheevi, diploid Aegilops speltoides acted as a maternal form. In addition to plasmatic genomes, this aegilops species introduced into them also the B and G nuclear subgenomes. A comparison of nucleotide sequences in the variable part of the promoter of evolutionary conserved rRNA genes in polyploid wheats with their counterparts in diploid wheats and aegilopses confirmed the accepted wheat phylogenies.

[The frequencies of the low-molecular-weight alanine-rich cold shock proteins genes and variation of their primary structure in plants of temperate climate]. / Rasprostranennost' genov nizkomolekuliarnykh alaninbogatykh belkov "kholodovogo shoka" v rasteniiakh umerennogo klimata i variabel'nost' ikh pervichnoi struktury.

Polymerase chain reaction (PCR) was used to study the frequencies of the gene of a low-molecular-weight alanine-rich cold shock protein in plants of temperate climate. This gene was demonstrated to be specific for the family Cruciferae. Its variability in this family was studied.

[Hydrocarbon-binding peptides from bean plant lectins in connection with various host specific macrosymbionts during development of symbiosis with rhizobium bacteria]. / Uglevodsviazyvaiushchie peptidy lektinov bobovykh rastenii v sviazi s razlichnoi khoziaiskoi spetsifichnost'iu makrosimbionta pri obrazovanii simbioza s kluben'kovymi bakteriiami.

The nucleotide sequences of 280-360-bp domains of lectin genes from 20 legume species belonging to 17 genera have been determined. A computer analysis of the sequences has been performed with the LASERGENE package. Based on this analysis, we constructed the phylogenetic tree of the lectins, which reflects their phylogenetic and evolutionary relationships, and predicted the amino-acid sequences of the corresponding protein domains. Features of the structure of the hydrocarbon-binding lectin domains were elucidated in some species of legume genera from the temperate climatic zone. The domains were highly variable and contained the consensus sequence AspTrePheXxxAsxXxxXxxTrpAspProXxxXxxIns/DelArgHis bearing the bulk of amino acid replacements, insertions, and deletions. An association between legume groups (including species from different genera and tribes) symbiotic with the same rhizobium species and the similarity between the hydrocarbon-binding domains of lectins from these plants was found.