[Proteomic Profile of the Bacterium Sinorhizobium meliloti Depends on Its Life Form and Host Plant Species].

The importance of root nodule bacteria in biotechnology is determined by their distinctive feature: symbiotic nitrogen fixation resulting in the production of organic nitrogen-containing compounds. While interacting with host legume plants, the cells of these bacteria undergo global changes at all levels of expression of genetic information leading to the formation in root nodules of so-called bacteroids functioning as nitrogen fixation factories. The molecular mechanisms underlying plant-microbial symbiosis are actively investigated, and one of the most interesting and poorly studied aspects of this problem is the species-specificity of interaction between root nodule bacteria and host plants. In this work we have performed the proteomic analysis of the Sinorhizobium meliloti bacteroids isolated from two legume species: alfalfa (Medicago sativa L.) and yellow sweet clover (Melilotus officinalis L.). It has been shown that the S. meliloti bacteroids produce a lot of proteins (many of them associated with symbiosis) in a host-specific manner, i.e., only in certain host plant species. It has been demonstrated for the first time that the levels of expression in bacteroids of the genes encoding the ExoZ and MscL proteins responsible for the synthesis of surface lipopolysaccha-rides and formation of a large conductance mechanosensitive channel, respectively, depend on a host plant species that confirms the results of proteomic analysis. Overall, our data show that the regulation of bacteroid development by the host plant has species-specific features.

HISTOLOGICAL CHANGES IN SPLEEN UNDER CONDITIONS OF TOXIC CARCINOGENESIS.

Under development of dimethylhydrazine-induced adenocarcinomatosis of the large intestine in white outbred male rats morphological changes of the structural components of the spleen were studied. It was found, that the progression of experimental carcinogenesis is accompanied by severe violations of the morphological state of all structural components of the spleen, manifested by destructively degenerative changes of the stroma, red and white pulp and significant vascular disorders. The severity of the pathomorphological changes in the spleen increases directly proportionally to the increase of the duration of the oncogenic factor impact.

Proteomic Analysis of Escherichia coli Protein Fractions Resistant to Solubilization by Ionic Detergents.

Amyloids are protein fibrils adopting structure of cross-beta spine exhibiting either pathogenic or functionally significant properties. In prokaryotes, there are several groups of functional amyloids; however, all of them were identified by specialized approaches that do not reveal all cellular amyloids. Here, using our previously developed PSIA (Proteomic Screening and Identification of Amyloids) approach, we have conducted a proteomic screening for candidates for novel amyloid-forming proteins in Escherichia coli as one of the most important model organisms and biotechnological objects. As a result, we identified 61 proteins in fractions resistant to treatment with ionic detergents. We found that a fraction of proteins bearing potentially amyloidogenic regions predicted by bioinformatics algorithms was 3-5-fold more abundant among the identified proteins compared to those observed in the entire E. coli proteome. Almost all identified proteins contained potentially amyloidogenic regions, and four of them (BcsC, MukB, YfbK, and YghJ) have asparagine- and glutamine-rich regions underlying a crucial feature of many known amyloids. In this study, we demonstrate for the first time that at the proteome level there is a correlation between experimentally demonstrated detergent-resistance of proteins and potentially amyloidogenic regions predicted by bioinformatics approaches. The data obtained enable further comprehensive characterization of entirety of amyloids (or amyloidome) in bacterial cells.

[Search for the genes critical for propagation of the prion-like antisuppressor determinant [ISP+] in yeast using insertion library].

The prion-like determinant [ISP+] manifests itself as antisuppressor of certain sup35 mutations. To establish that [ISP+] actually represents a new yeast prion, it is necessary to identify the gene encoding protein corresponding in its prion form to [ISP+]. Analysis of transformants obtained by transformation of [ISP+] strain with insertion gene library revealed three genes controlling the [ISP+] maintenance. These genes are UPF1, UPF2 and SFP1. The SFP1 encodes potenlially prionogenic protein, as it is enriched with asparagine and glutamine residues. Therefore it is the most likely candidate to the role of [ISP+] structural gene. The UPF1 and UPF2 products are components of nonsense-mediated mRNA decay. It was shown that [ISP+] elimination caused by UPF1 and UPF2 inactivation is reversible. It was shown also that Upf1 and Upf2 proteins are not related functionally to Ppzlp phosphatase, influencing [ISP+] manifestation. Possible mechanisms of UPF1 and UPF2 influence on [ISP+] maintenance are discussed.

[Phenotypic manifestation of epigenetic determinant [ISP+] in Saccharomyces serevisiae depends on combination of mutations in SUP35 and SUP45 genes].

It is known that translation fidelity in Saccharomyces yeast is determined by factors of genetic and epigenetic (prion) nature. The work represents results of further analysis of strains containing non-chromosomal determinant [ISP+], described earlier. This determinant is involved in the control of translation fidelity and some of its properties indicate that it is a prion. [ISP+] manifests phenotypically as antisuppressor of two sup35 mutations and can be cured by guanidine hydrochloride (GuHCl). Here we have shown that sup35 mutants containing [ISP+] contain also additional sup45 mutations. These mutations cause amino acid replacements in different regions of eRF1 translation termination factor, encoded by SUP45 gene. Strains bearing sup35-25 mutation contain sup45 mutation, which causes amino acid replacement at position 400 of eRF1; strains bearing sup35-10 contain mutation causing replacement, which alters eRF1 at position 75. Thus, antisuppressor phenotype of [ISP+] strains depends on interaction of sup35 and sup45 mutations, as well as on the GuHCl-curable epigenetic determinant.

[Characterization of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1]. / Kharakteristika missens-mutatsii v gene SUP45 drozhzhei Saccharomyces cerevisiae, kodiruiushchem faktor terminatsii translatsii eRF1.

Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1-eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.

[Interaction of ATP17 gene with SUP45 and SUP35 genes in Saccharomyces cerevisiae yeast]. / Vzaimodeistvie gena ATP17 s genami SUP45 i SUP35 u drozhzhei Saccharomyces cerevisiae.

Genes SUP35 and SUP45 have been identified in the saccharomycete yeast as genes controlling termination of translation in cytoplasmic ribosomes. However, many facts indicate that the control of translation termination is not the only function of these genes. This work is devoted to studying one of the pleiotropic effects of sup35 and sup45 mutations, a respiratory deficiency. The compensation for this deficiency in mutants for either gene can occur due to a mutation in the ATP17 gene encoding the f-subunit of mitochondrial F1F0 ATP synthase. It is assumed that the observed interaction can be related to the system of co-translational protein import into mitochondria.

[Polymorphism of the SUP35 gene and its product in the Saccharomyces cerevisiae yeasts]. / Polimorfizm gena SUP35 i ego produkta u drozhzhei Saccharomyces cerevisiae.

The product of the SUP35 gene of the saccharomycete yeast, the translation termination eRF3 factor, can be converted in prion, the heritable determinant of protein nature. The nucleotide sequence of this gene from the strain belonging to Peterhof genetic lines of the yeast Saccharomyces cerevisiae was determined. A comparison of the identified sequence with SUP35 sequences in the database of GenBank allowed the detection of polymorphic sites both in the SUP35 gene and its product. The location of polymorphic sites in the evolutionarily nonconserved N-terminal protein region confirmed that this eRF3 fragment lacks functions vital to life activity. Nevertheless, these sites are located in the vicinity of sites, whose role in the prion conversion of eRF3 has been established. Based on this, natural polymorphism of the primary eRF3 structure is assumed to be connected with the existence of different variants (strains) of its prion analog.

Isolation and characterization of myeloperoxidase from leukocytes of rat peritoneal fluid.

Myeloperoxidase (MPO) was isolated from rat peritoneal leukocytes with a yield of 51% and A430/A280 = 0.75 - 0.80, and its physicochemical properties were studied. The molecular weight of the MPO is about 150 kD. The MPO was assayed for amino acid content. We used substrate mixture containing phenol, 4-aminoantipyrine, and H2O2 to detect 10(-10) M of the enzyme. The MPO was localized in rat blood neutrophils using polyclonal anti-MPO antibodies and secondary fluorescein isothiocyanate-labeled antibodies. Immunofluorimetric assay (IFMA) was developed for quantitative measurement of the MPO. The MPO and leukocytes can iodinate BSA using NaI or thyroxine as the source of iodine.