Exotoxin diversity of Staphylococcus aureus isolated from milk of cows with subclinical mastitis in Central Russia.

Mastitis, a major veterinary problem widespread in many regions, is caused mainly by Staphylococcus spp. However, there is no current reliable information about the role of Staphylococcus aureus and their toxins in the development of mastitis in cows in the territory of the Russian Federation. The aim of this investigation was to determine the profile of exotoxins of S. aureus from cow milk from farms of Central Russia. A total of 60 isolates of S. aureus were obtained from milk samples of cows with the subclinical form of mastitis. The exotoxin genes were identified using 2 types of PCR assays. The diversity of enterotoxin genes was studied by multiplex PCR. The percentage occurrence of enterotoxin genes was as follows: sea, 53.3%; seb, 3.3%; sec, 50%; sed, 4%; see, 46.6%; seg, 70%; sei, 10%; selp, 3.3%; and tsst1, 1.6%. The seh gene was not detected. The genes of pore-forming toxins and phenol-soluble modulins were identified by singleplex PCR and consisted of the following: hlA, 70%; lucS, 46.6%; psmA, 81.6%; psmB, 95%; and hld, 78.3%. The most abundant genes were psm (psmB, 95%), which codes for pore-forming toxins, and seg (70%), which codes for enterotoxins. The production of some enterotoxins in bacterial culture medium was detected by ELISA. The level of toxin production was near 1 ng/mL for SEA, SEE, SEG, SEI, SELP, and TSST-1 and reached a maximal level of 18 ng/mL for SEE. In the present work, we show that subclinical mastitis in cows is associated with S. aureus in the central region of the Russian Federation. Most of the isolates containing enterotoxin genes also had cytotoxin genes.

Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1.

The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling.

Search for ligand of N-acetylglucosaminyl-N-acetylmuramyl dipeptide using its peptide mimetic.

The method for searching for ligands exerting an adjuvant effect is described. The method involves isolation of polysomes using an immobilized peptide mimetic of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) - RN-peptide. After the affinity chromatography and washing, RN-peptide complexes with the target sequences were dissociated with guanidine hydrochloride. The obtained mRNA was used for cDNA synthesis and subsequent cloning in an expression vector. Further studies showed the effectiveness of this method. Clones interacting with the peptide were selected using biotinylated RN-peptide. It was found that all clones encode a sequence identical to the protein YB-1. Recombinant antibodies against protein YB-1 were selected from a phage display human scFv library. Using these antibodies, we determined the binding constant of RN-peptide to protein YB-1. Competitive analysis showed that RN-peptide and GMDP compete for the same portion of YB-1 at molar ratio 1 : 12.

Effect of the format of antibodies on their specificity.

The influence of alterations in the format of antibodies on their specificity has been examined. To analyze the role of Ig constant regions in recognizing antigens, a comparison was made of the specificities of full-scale murine monoclonal antibodies and scFv single-chain miniantibodies obtained from the latter with regard to a group of closely related protein antigens - Staphylococcus enterotoxins. It was found that in the scFv format the specificity and affinity of miniantibodies diminished as compared to the full-scale ones. Specificity of antibodies may be enhanced by transforming them into full-scale antibodies. Moreover it was shown that miniantibodies within a phage particle generated from combinatorial phage libraries possess greater specificity to the antigen, however during the subsequent transformation to soluble scFv antibodies their specificity diminishes.

Structural modification effects on bioactivities of the novel 15-mer peptide adjuvant.

Structure-function relationship studies for novel GMDP-peptide mimic, RVPPRYHAKISPMVN (RN-peptide) were performed on array of truncated and 'Ala-scan' analogues. The shortest peptide fragment possessing detectable affinity towards anti-GMDP-antibodies was demonstrated to be PRYH. RN-peptide analogues lacking up to 8 residues at C-terminus were found to retain adjuvant activity with the minimal active peptide being RVPPRYH. Evaluation of Ala-scan analogues highlighted that adjuvant activity is most critically dependent on both arginine residues, but also is sensitive to substitution of K9, I10, S11 and M13 amino acid residues, the functional importance of which was additionally confirmed by testing peptides truncated at both termini.

Simultaneous detection of seven staphylococcal enterotoxins: development of hydrogel biochips for analytical and practical application.

A method of simultaneous analysis of staphylococcal enterotoxins using hydrogel-based microarrays (biochips) has been developed. The method allows simultaneous quantitative detection of seven enterotoxins: A, B, C1, D, E, G, and I in a single sample. The development of the method included expression and purification of recombinant toxins, production of panels of monoclonal antibodies (mAbs) against the toxins, and design and manufacturing of an experimental biochip for the screening of mAbs and selection of optimal pairs of primary and secondary antibodies for each toxin. The selected mAbs have high affinity toward their targets and no cross-reactivity with unrelated enterotoxins. Finally, a diagnostic biochip was designed for quantitative analysis of the toxins, and the analytical protocols were optimized. The sensitivity of the detection reached 0.1-0.5 ng/mL, depending on the type of enterotoxin. The evaluation of the resulting biochip using spiked food samples demonstrated that the sensitivity, specificity, and reproducibility of the proposed test system fully satisfy the requirements for traditional immunoanalytical systems. The diagnostic biochips manufactured on reflecting metal-coated surfaces shortened the time of analysis from 17 to 2 h without loss of sensitivity. The method was successfully tested on samples of food and biological media.

[A method for the preparation of adjuvant peptide mimetics of GMDP with the use of monoclonal antibodies and combinatorial libraries of peptides in the format of phage display].

A method for the preparation of peptide mimetics of GMDP which could exhibit adjuvant activity without the negative effects of GMDP is described. The search for peptides with GMDP-like adjuvant activity was performed using highly specific monoclonal antibodies against GMDP and combinatorial peptide libraries in the format of phage display. Various elution methods were used for the immunoaffinity enrichment of the libraries during the course of the preparation of highly active and specific peptides. A sole peptide (Arg-Val-Pro-Pro-Arg-Tyr-His-Ala-Lys-Ile-Ser-Pro-Met-Val-Asn, RN) was obtained by the elution of phage particles from the immunosorbent with a 1 -microM solution of the natural ligand (GMDP). Elution with a buffer with a low pH value (0.1 M glycine-HCl, pH 2.2) gave two other peptides: Ser-Gly-Arg-Val-Ala-Val-Ser-Pro-Asp-Ser-Pro-Leu-Phe-Tyr-Pro (SP) and Arg-Tyr-Gly-Gly-Ser-Val-Leu-Asn-Ile-Glu-Cys-Gln-Phe-Tyr-Gly (RG). Affinity constants for the RN and SP peptides proved to be 3.6 x 10(8) and 3.5 x 10(8) M(-1), respectively. The specificity of the interaction with the monoclonal antibodies was checked by the competitive displacement of the peptides from the antigen-antibody complex by GMDP. The RN peptide exhibited adjuvant activity similar to that of GMDP, but had no pyrogenic effect characteristic of GMDP. The described method could be used for the search for mimetics of biologically active low-molecular compounds.

Refolding of scFv mini-antibodies using size-exclusion chromatography via arginine solution layer.

The method for refolding of mini-antibodies using size-exclusion chromatography via arginine solution layer was developed. This method allows to refold scFv, to separate both aggregated protein and low molecular weight compounds and to isolate functionally active protein preparation in monomeric form. The comparison of various scFv preparations isolated either from inclusion bodies or from soluble fraction revealed that refolded mini-antibodies demonstrate higher antigen-binding activity. Mini-antibodies refolded in the presence of arginine also demonstrate higher electrophoretic mobility during native PAGE in comparison with soluble cytoplasmic antibodies. Both soluble as well as refolded antibodies had similar CD spectra. Refolded mini-antibodies are storage-stable.

[Construction of combinatorial immune library of single chain human antibodies to orthopoxviruses and selection from this library antibodies to recombinant protein prA30L of variola virus].

A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.

[Development of recombinant scFv-antibodies against diphtheria toxin using phage display system].

Phage display technology is an effective approach to the development of the next generation of immunodiagnostic reagents. Naive murine phage display a library of single-chain variable antibodies (scFv) was used to isolate scFv recognizing the diphtheria toxin, an important diagnostic antigen of diphtheria. The diphtheria toxin B subunit-binding clone with affinity constant of 1.13 x 10(7) M(-1) was selected. scFv preserved activity on storage in the course of 8 months.

Epitope mapping of human Fas using peptide phage display.

Sandwich EIA for measurement of soluble Fas was developed on the basis of SA-7 and SA-8 monoclonal antibodies to full-length human Fas. The threshold sensitivity of the test system is 0.3 ng/ml. Several isoforms of soluble Fas were identified. The structure of SA-7 and SA-8 antibody epitopes was determined using the peptide phage library. It was shown that SA-7 antibody epitope is determined by amino acid residues 129-134 (CKPNFF), while SA-8 antibody epitope is determined by amino acid residues 94-99 (KAHFSS) of full-length Fas. Hence, sandwich EIA on the basis of SA-7 and SA-8 monoclonal antibodies for detection of soluble Fas in human serum is to detect the following Fas isoforms: FasExo6Del, FasExo4Del, FasExo4,6Del, FasExo4.7Del, and FasExo8Del.

[Construction and characterization of combinatorial naive phage library of human scFvs].

A combinatorial phage display library of human single-chain antibody fragments (scFv) was constructed on the basis of variable domains of heavy (Vh) and light (VI) genes cloned from the lymphocytes of six healthy donors. The size of the library was 2? 10(8) independent clones. Single-chain antibodies against recombinant human TNF?, vaccinia virus and virus-like particles formed by core protein of hepatitis B virus were selected from the library. Unique scFv sequences were identified using the HaeIII fingerprinting. The specificity of the selected clones was proved by the Western-blot analysis.

Selection and characterization of phage miniantibodies to actins of different origin.

Single-chain miniantibodies (scFv's) to actin were obtained by the phage display method. A naive combinatorial phage display library of murine scFv's (containing 2x10(8) independent recombinant clones) was used to select miniantibodies. After three rounds of selection two clones producing miniantibodies to chicken smooth muscle actin with affinity constants of 1.4x10(7) and 1.2x10(6) M(-1) were chosen. The isolated miniantibodies could specifically detect various plant and animal actins.

[The production of mini antibodies against human granulocyte colony-stimulating factor using murine scFv combinatorial library]. / Poluchenie mini-antitel protiv granulotsitarnogo koloniestimuliruiushchego faktora cheloveka s ispol'zovaniem biblioteki scFv myshi.

To develop a phage display of single-chain antibodies (scFv), fractions of total cell DNA and RNA were obtained from splenocytes of naive mice. The DNA fragments encoding variable regions of light and heavy immunoglobulin chains were amplified and isolated using primers specific to the conservative regions of these genes. The construction of the library was based on the principle of stochastic combining of the DNA fragments encoding the light and heavy antibody chains with the DNA linker, whose structure corresponded to the (Gly4Ser)3 sequence. The scFv library was constructed using the E. coli TG1 strain and the phagemid vector pHEN1. The repertoire of the library exceeded 5 x 10(7) independent recombinant clones. The clones producing antibodies to the granulocyte colony-stimulating human factor were isolated. The affinity constants of the resulting scFv were in the range of 2 x 10(4) to 1.8 x 10(7) M-1.

Subtractive hybridization of biotinylated DNA in phenol emulsion.

A method of subtractive hybridization using biotinylated DNA and phenol emulsion reassociation technique (PERT) has been proposed. A possibility of combining these techniques has been shown for the first time. The effect of biotinylation degree on the formation of water-insoluble Bio-DNA aggregates was studied. The conditions when Bio-DNA aggregation is actually absent were revealed. A possible use of the above method in hybridization experiments in a wide range of DNA concentrations has been shown. The time of hybridization was 0.5-1 h. The method was approved on a model system, and its possible application for the enrichment of rare mRNA was shown. No less than 300-fold enrichment is achieved for a rare transcript (IL-2) in three cycles of subtractive hybridization.

[Facultative and obligate aerobic methylobacteria synthesize cytokinins]. / Fakul'tativnye i obligatnye aérobnye metilobakterii sinteziruiut tsitokininy.

The presence and expression of genes controlling the synthesis and secretion of cytokinins by the pink-pigmented facultative methylotroph Methylobacterium mesophilicum VKM B-2143 with the serine pathway and nonpigmented obligate methylotroph Methylovorus mays VKM B-2221 with the ribulose monophosphate pathway of C1 metabolism were shown using the polymerase chain reaction (PCR) and reverse transcription-PCR methods. The presence of the corresponding mRNA in M. mesophilicum cells grown on methanol or succinate suggests that the expression of these genes is constitutive. The cytokinin activity of culture liquid and its fractions was determined by a biotest with Amarantus caudatus L. seedlings. Using enzyme-linked immunosorbent analysis, we detected zeatin (riboside) in the culture liquid of both bacteria studied. The data obtained show that the aerobic methylobacteria are phytosymbionts that are able to utilize the single- and polycarbon compounds secreted by symbiotic plants and to synthesize cytokinins.

[The spatial-temporal distribution of the mRNA of the Na+-K+-ATPase alpha-subunit in the early development of the clawed toad studied by hybridization in situ]. / Izuchenie prostranstvenno-vremennogo raspredeleniia mRNK al'fa-sub''edinitsy Na+-K+-ATFazy v rannem razvitii shportsevoi liagushki metodom gibridizatsii in situ.

Distribution of Na+-K+-ATPase alpha-subunit mRNA in full-grown oocytes and early embryos from of X. laevis has been studied using in situ hybridization on histological sections. The mRNA is dispersed about equally in the cytoplasm and is absent from the nucleus. The concentration of the mRNA is successively decreased from fertilization to the late blastula. The drastic decrease in mRNA concentration is observed in a period from morula to mid-blastula. The local increase in the mRNA concentration was detected in dorsal mesoderm and ectoderm during gastrulation.