Phylogenetic Analysis and Molecular Typing of Trichothecene-Producing Fusarium Fungi from Russian Collections.

We performed a three-locus phylogenetic analysis of Fusarium strains presumably capable of trichothecene production, which were deposited in the Russian national collections. The intra- and interspecific polymorphism of partial sequences of the translation elongation factor 1 alpha (TEF1α) gene and two genes from the trichothecene cluster TRI5 and TRI14 was studied. A study of 60 strains of different origins using DNA markers confirmed, and in the case for several strains, clarified their taxonomic characteristics. As a result, a strain of F. commune (F-900) was identified in Russia for the first time. Furthermore, the strain F-846 proved to be phylogenetically distinct from any of the known Fusarium species. F. equiseti strains from Northwest Russia were found to belong to the North European group (I), whereas a strain from the North Caucasus - to the South European one (II). Partial TRI14 sequences from 9 out of 12 species were determined for the first time. Their comparative analysis demonstrated a relatively high level of intraspecific variability in F. graminearum and F. sporotrichioides, but no correlation between the sequence polymorphism and the geographic origin of the strains or their chemotype was found. Specific chemotypes of trichothecene B producers were characterized using two primer sets. The chemotyping results were verified by HPLC.

[Production and characteristics of monoclonal antibodies to the diphtheria toxin].

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

[Preparation and characterization of monoclonal antibodies to the cholera toxin].

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.

[Molecular cloning, expression, and characterization of human mini-antibodies against enterotoxin C1 of Staphylococcus aureus].

We describe here the cloning, expression, and production of specific single-chain antibodies (scFv) against the recombinant enterotoxin C1 of Staphylococcus aureus. High-affinity scFv were selected from the phage library of human mini antibodies; afterwards, the cells of E. coli trxA gor double mutant were infected with a product obtained by fusion of DNA encoding of these mini antibodies with the trxA gene to induce soluble scFv synthesis in cell cytoplasm. The scFv obtained displayed high enterotoxin C1 affinity. Analysis for cross reactivity showed that mini-antibodies interacted also with SEA- SEB-, SED-, SEE-, SEG-, and SEI-type enterotoxins, but they failed to interact with ricin, diphtheritic, and cholera toxins, or with both lethal and protective factors of the anthrax toxin. This property may be helpful in screening for staphylococcus enterotoxins.

[Isolation and characterization of the ALP1 protease from Aspergillus fumigatus and its protein inhibitor from Physarium polycephalum].

It is known that Aspergillus fumigatus secretes a serine protease ALP1 of the subtilisin family in the presence of extracellular protein substrates. We found conditions of A. fumigatus culturing that provide a high ALP1 activity inside cells without induction by extracellular proteins. The identity of the properties of the secreted and intracellular enzymes was shown. A thermostable protein inhibitor of the ALP1 protease was isolated from the plasmodium of the myxomycete Physarum polycephalum. Its molecular mass is 32-33 kDa. The inhibitor inhibits the ALP1 protease activity with IC50 of 0.14 microM. This protein was also shown to be a less efficient inhibitor of the activity of HIV-1 protease (IC50 2.5 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

[Bacteriorhodopsin retains the conformation of Val69-Gly72 fragment during functioning]. / Sokhranenie konformatsii fragmenta Val69-Gly72 bakteriorodopsina pri ego funktsionirovanii.

Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of functioning bacteriorhodopsin molecule. Using the methods of solid phase enzyme immunoassay, peptide phage display, and 1H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4, which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.

[Polypeptides from murine peritoneal macrophages recognizing glycosaminylmuramoyl dipeptide]. / Polipeptidy iz peritoneal'nykh makrofagov myshi, uznaiushchikh gliukozaminilmuramoildipeptidy.

By means of radioligand analysis, murine peritoneal macrophages were shown to express several hundreds cell surface high-affinity GMDP-binding sites with a binding constant 350 pM. Photoaffinity labeling followed by SDS-PAGE enabled us to identify inside these cells 32-34 and 38 kDa proteins, specifically binding GMDP. Proteins 32-34 kDa were also detected by Western blotting analysis using biotinylated conjugate of polyacrylamide with immobilized GMDP-Lys [(GMDP-Lys)-PAA-(Bi)] in cell lysate of murine peritoneal macrophages.

Biochemical characterization of glucosaminylmuramyldipeptide binding sites of murine macrophages.

By using radioligand analysis, murine peritoneal macrophages were shown to express several hundred high-affinity cell surface GMDP-binding sites (Ka 350 pM). Photoaffinity labeling followed by SDS-PAGE enabled us to identify 32-34 and 38 kDa proteins inside these cells that bound GMDP specifically.

Bispecific monoclonal antibodies to human interleukin 2 and horseradish peroxidase.

Hybrid hybridomas (tetradomas), producing bispecific monoclonal antibodies (bmabs) binding to both interleukin 2 and horseradish peroxidase were obtained by fusing IL 2-specific and HRP-specific hybridomas. Parental hybridomas were labelled prior to the fusion with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate. Bifluorescent (tetradoma) cells were sorted out using fluorescence activated cell sorter. Two clones, designated D8C1/G and H7C11/H were shown to secrete bmabs over at least 6 months growth in culture. Bmabs have been purified from mouse ascites by ammonium sulfate precipitation and affinity chromatography on immobilized peptide, modelling corresponding IL 2 epitope. The purity of antibodies obtained was characterized by capillary electrophoresis. The possibility of using these antibody preparations in rIL 2 analysis was evaluated in two types of EIA: direct and competitive solid-phase EIA. Both assays had similar sensitivity of about 1.5-4 ng IL 2 per ml.

[Conformation of fragment 66-72 of interleukin-2 complexed with a monoclonal antibody to interleukin-2]. / Konformatsiia fragmenta 66-72 interleikina-2 v komplekse s monoklonal'nym antitelom k interleikinu-2.

1H-NMR spectra of the interleukin-2 synthetic fragment Ac-Leu66-Glu-Glu-Val-Leu-Asn-Leu72-OCH3 in the presence or absence of the monoclonal antibody were analysed. The data obtained are consistent with an extended unordered conformation of the free peptide. Measurements of NOESY cross-peak intensities allowed us to determine the spatial structure of the peptide bound to the antibody. The peptide has an amphiphilic surface with hydrophobic and hydrophilic amino acid side chains clustered on the opposite sides of its alpha-helical-like structure. The hydrophobic and hydrophilic clusters are located on the opposite sides of the bound peptide's surface. The hydrophobic side chains contact the antibody surface, while the hydrophilic ones are oriented into the solvent (T. A. Balashova et al. (1991) Bioorgan. Khim. (USSR), v. 17, p. 1470-1486). Hydrolysis of the methyl ester slowly ocurs in the presence of the antibody. This process does not alter the conformation of the peptide bounded with the antibody, though decreases the peptide's affinity to the antibody.