Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

[Unique steroid 21-hydroxylase gene CYP21A2 polymorphism in patients with hyperandrogenism signs].

Hyperandrogenism is a medical condition characterized by excessive production of male sex hormones (androgens) in woman organism. One of the major causes of hyperandrogenism is the autosomal-recessive disorder--congenital adrenal hyperplasia (CAH). The mutational defects in the steroid 21-hydroxylase CYP21A2 gene causing steroid 21-hydroxylase deficiency account for over 90% of CAH cases. Our paper describes the sequencing results of entire CYP21A2 gene from 15 patients with hyperandrogenism signs, which had not nine most prevalent mutations associated with nonclassic CAH as it was previously established. 26 polymorphisms were found by sequencing among which 25 were known previously and 23 of them are referred to "normal" gene variants which do not associated with CAH. At the same time the gene of every patient had unique its own distinctive combination of polymorphisms. New SNP represents synonymous substitution C --> T in 3' part of exon 8. All detected SNPs are not regularly distributed but are clustered along the gene. Notably, they were found in the neighborhood of initiation and termination codons and near the intron-exon boundaries of introns 2, 6 and 8. We hypothesize that "normal" clinically insignificant per se SNPs in unique combinations may influence spatial structure of CYP21A2 mRNA or its pre-mRNA splicing efficiency and decrease gene expression level. This assumption may explain the mechanism of pathological phenotype development in our patients.

[Preparation and characterization of the recombinant protein containing immunomimetic peptide of benzo[a]pyrene].

Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.

The study of the interaction of (cytosine-5)-DNA methyltransferase SsoII with DNA by acoustic method.

The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5'-CCNGG-3' (N=A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5'-AGGACAAATTGTCCT-3' in the promoter region of SsoII restriction-modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, K(D), of the M.SsoII-DNA complexes decreased in the order 60oct>60reg>60met, suggesting a higher stability of M.SsoII-60met complex in comparison with the others. The association rate constant, k(a), was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell.

Chimeric lactase capable of spontaneous and strong immobilization on cellulose and development of a continuous-flow system for lactose hydrolysis at high temperatures.

Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.

[A new DNA diagnostic system for the detection of human CYP21 gene mutations associated with adrenal cortex hyperplasia].

Congenital Adrenal Hyperplasia (CAH) is one of the most widespread severe autosomal recessive hereditary diseases. CAH is caused by the impaired biosynthesis of the key human hormones cortisol and aldosterone and is accompanied by the excess synthesis of androgens. Over 90% of CAH cases are caused by a deficiency of the steroid 21-hydrohylase (P450c21). The degree of damage in this enzyme is responsible for the severity of the clinical manifestation of CAH from potentially lethal to mild symptoms. Various mutations of the gene encoding this enzyme are the main source of the reduced activity of the 21-hydrolase. The location of the highly homological pseudogene CYP21P in close proximity to the functional gene impedes the DNA diagnostics of CAH. To detect the eight most frequent CYP21 gene mutations associated with CAH, we developed a new real-time PCR-based system of DNA diagnostics using new allele-specific primers and TaqMan probes for the analyzed mutations. The method was primarily tested on artificial DNA templates, where the analyzed mutations were introduced by site-directed mutagenesis. Then, it was tested on DNA samples from 43 patients with clinical and biochemical manifestations of CAH; seven patients were used as a control. Two mutant alleles were detected in two different individuals: the nonsense Q318X and the missense V281L mutations.

[Feasibility to use of recombinant domains of immunoglobulin-like protein LigA as a promising marker for serological testing of patients with leptospirosis].

AIM: To clone the DNA fragment encoding conservative domain of LigA protein of Leptospira interrogans into Escherichia coli and to investigate antigenic properties of constructed chimeric protein. MATERIALS AND METHODS: E. coli strain M15 [pREP4], recombinant plasmid pTT10 encoding cellulose-binding domain (CBD), restriction endonucleases BamHI, BglI, BglII, XbaI, T4 DNA-ligase, RNAse were used in the study. Molecular cloning of ligA gene fragment was performed using standard protocols, and expression of hybrid genes--according to "Qiagen company's protocols. Extraction and purification of proteins were performed using original method. RESULTS: DNA fragment encoding immunoglobulin-like domain 5 of LigA was cloned in E. coli. Effective strain-producer of chimeric domain D5-CBD consisting of the immunoglobulin-like domain 5 of LigA, Gly-Ser spacer, and cellulose-binding domain (CBD) was obtained. The high-purity D5-CBD preparation was obtained using one-stage purification on cellulose. Antigenic specificity of this chimeric protein was studied and it was shown that it could be used as a marker for the development of diagnostic ELISA kit. CONCLUSION: Recombinant domain of LigA in chimeric protein produced in E. coli retains antigenic properties of native LigA protein. Obtained results confirm the feasibility to use recombinant antigen D5-CBD as a marker for development of diagnostic kits on the basis of ELISA.

[Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo].

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.

[Isolation and characterization of collagen-binding domains from human von Willebrand factor].

DNA fragments encoding for two collagen binding decapeptides from the human von Willebrand factor (vWF-H1 and vWF-H2) were cloned in the Escherichia coil culture. Overproducing strains of the chimeric proteins vWF(H1)-CBD and vWF(H2)-CBD consisting of the corresponding decapeptide, Gly-Ser spacer and a cellulose binding domain (CBD) from Anaerocellum thermophilum were constructed. Using one-stage purification on cellulose, the highly purified samples of vWF(H1)-CBD and vWF(H2)-CBD proteins were obtained and the ability of these proteins to bind collagen was studied. These constructions are planned to be used for development of the recombinant collagen binding proteins with different biological activities, which, in its turn, will be used for development of the new generation products and materials for medicine, such as different kinds of implants, the coats, etc.

[Research into the immunogenic properties of the recombinant cellulose-binding domain of Anaerocellum thermophilum in vivo].

Development of new technology allows different antigens of a necessary degree of cleanliness to be obtained. This development is a major problem of modern medical biotechnology. A promising approach to this problem includes use of the affinity domains (tags) incorporated in structure of a recombinant antigen and capable to bind to corresponding sorbents. The method of preparation of ready-for-use injections containing complexes formed by soluble antigens on insoluble cellulose immunosorbent (not chemical conjugates) in one stage is based on the fusion protein technology. This approach includes preparation of two-component recombinant proteins containing an antigen of interest and the cellulose-binding domain (CBD), which spontaneously binds to cellulose containing sorbents with high binding constant. Research into the immunogenic properties of the CBD in the complex with cellulose and in the preparation of recombinant CBD in a rat model was performed. The titers of specific antibodies in rat serum induced by recombinant CBD and CBD in the complex with cellulose was evaluated. The CBD in the complex with cellulose was more immunogenic in comparison with CBD alone. The spectrum and levels of cytokines in collected rat serum induced by developed preparations was also measured using the microsphere-based Luminex Flowmetrix system (BioPlex). It was found that the amorphous cellulose was not an immunotolerant sorbent, because it induced the expression of the proinfammatory cytokines in vivo.

SsoII-like DNA-methyltransferase Ecl18kI: interaction between regulatory and methylating functions.

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Delta(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyltransferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.

[Immunization with cellulose-immobilized antigens. The development of A. E. Gurvitch concept].

A highly purified TUL4-CBD chimeric protein was obtained by one stage purification method. TUL4-CBD protein consists of TUL4 Francisella tularensis mature peptide sequence, Gly-Ser spacer and cellulose binding domain (CBD) of Anaerocellum thermophilum. The TUL4-CBD protein was shown to induce production of specific antibodies to TUL4 protein in laboratory animals.

[The flower and blossom morphology of Asteraceae correlates with composition of their pollinators]. / Sviaz' mezhdu stroeniem tsvetkov i sotsvetii slozhnotsvetnykh (Asteraceae) i sostavom ikh opylitelei.

The correlation between flower morphology and share of different insect groups visiting them was studied for 15 Asteraceae species. We measured length and width of corolla tube of 100 flowers of each plant species and determined proportions of main groups of anthophilous insects during all blooming period. According to corolla length species under study ranged more or less uniformly from 2.16 mm (Tripleurospermum inodorum) up to 21.06 mm (Cirsium heterophyllum). The correlation between share of long-tongued bees (mainly bumblebees) among all visitors of inflorescens and corolla length was positive (r = 0.737, P < 0.01) while for short-tongued flies (Syrphidae, Muscidae, Calliphoridae) it was negative (r = -0.869, P < 0.01). It is interesting, that the point of crossing of regression lines (12 mm) approximately coincides with change in inflorescences coloration. Plants with corolla length less than 10 mm have yellow or white inflorescences that are visited primarily by flies, while the plants with longer corolla have violet or dark blue inflorescences, by bumblebees. The dependence of proportion of short-tongued solitary bees (Andrenidae, Halictidae) on a corolla length was non-linear. It increased with increase in corolla length in an interval of 2.16-6.26 mm (r = 0.930, P < 0.1), but decreased for longer corollas (r = -0.680, P < 0.05). The correlation between corolla length and proportions of beetles and butterflies were insignificant.

[Covalent binding of Cys142 from SsoII methyltransferase with DNA duplexes, containing a phosphoryldisulfide internucleotide group]. / Kovalentnoe sviazyvanie Cys142 metiltransferazy SsoII s DNK-dupleksami, soderzhashchimi fosforildisulfidnuiu mezhnukleotidnuiu grupppu.

DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.

[Structure of coadaptive complex of forest entomophylous plants with broad spectrum of pollinators]. / Struktura koadaptivnogo kompleksa lesnykh éntomofil'nykh rastenii s shirokim krugom opylitelei.

Pollinators of common forest entomophylous plants were studied at 1982-1985 near Zvenigorod biological station (Moscow region) and at 1989-1993 near Torma (Jogeva maakond, Estonia). The comparative analysis of spectra of pollinators has allowed to distinguish five groups (subcomplexes) of plants characterized by dominance of different groups of pollinators: myiophylous (flies from superfamily Muscoidea dominate), syrphidophylous (flies from family Syrphidae dominate), nonspecialized melittophylous (Apoidea, mainly bumblebees, dominate), psychophylous (butterflies dominate), and cantharophylous (beetles dominate). The belonging of plants to concrete subcomplex is determined by the morphology of flowers and influorescences, but in some cases habitat and time of blooming are also important. From year to year the composition of pollinators of the same plant species varies because of change in abundance of different groups of pollinators. However for long term these fluctuations are leveled off. External reproductive isolation of plants of myiophylous subcomplex is achieved mainly by spatial (ecological) and time (different time of flowering) isolations. However when two species of the same subcomplex grow together and their flowering time strongly overlap, there are some distinction in their pollinators reducing competition for pollinators.

[Comparison of imaginal diets of various Syrphidae (Diptera) species]. / Sravnenie imaginal'nogo pitaniia nekotorykh vidov zhurchalok (Diptera, Syrphidae)

The full amount and species composition of pollen from intestines of 11 species of Syrphidae (Arctophila fulva, Eristalis arbustorum, E. nemorum, E. pertinax, Helophilus pendulus, Myiatropa florea, Rhingia campestris, Sericomyia silentis, Syrphus baltheatus, S. ribessii and S. vitripennis), collected at the some place (Torma, Jogeva distr., Estonia) and at the some time (18-20 July 1989) have been studied. Maximum number of pollen grains is different fly species varied from 67,800 (Rh. campestris) up to 240,700 (S. silentis) grains, and average number from 25,560 (Rh. campestris) up to 115,880 (E. pertinax) grains. Maximum volume of pollen in different fly species varied from 1.5 (S. ribesii) up to 23.6 (S. baltheatus) mm3, and average volume from 0.36 up to 7.0 mm3 (the same species). The difference in a imaginal diets of Syrphidae are found, and the degree of difference does not correlate with a degree taxonomic affinity of species. The difference in strategy of a feeding behavior of two Syrphus species, that have similar diet, are marked: in intestines of 80% specimens of S. ribesii we found pollen grains of less that 7 plant species, whereas intestines of more than 88% specimens of S. vitripennis contained more than 8 species of grains. Distinction in alimentary preferences of different species of files can not be explained neither particularities of their morphology, nor their color preferences.

[The mechanisms of the restriction of pollinator range in the fireweed (Chamaenerion angustifolium) and 2 species of geranium (Geranium palustre) and G. pratense)]. / Mekhanizmy ogranicheniia kruga opylitelei u ivan-chaia (Chamaenerion angustifolium) i dvukh vidov geranei (Geranium palustre i G. pratense).

Flowers of Chamaenerion angustifolium, Geranium palustre and G. pratense are visited by more than 100 insect species. For all plants the spectrum of visitors is similar. However the role of insects species in pollination is different and depends on the stamen and stigma length, flexibility of pedicle and feeding behaviour of insect inside flower. The possibility to take and to transport pollen grains usually increase with the size of insect. The nature of this correlation is determined by stamen and stigma length. Pollinators of G. palustre with short stamens and stigmas are smaller than those of Ch. angustifolium and G. pratense. On the other hand, more flexible pedicle of G. palustre prevent the flowers from visits of big insects. Three plants studied by the authors are not equally attractive for different insect groups. Dense in fluorescence of Ch. angustifolium and G. pratense that usually are lifter under the grass are very attractive for foraging social insects (honey bee, bumblebee). Flies avoid long distance travelling and prefer single flowers located not far from each other. For instance G. palustre is more attractive for flies not for social bees. It is pollinated mainly by flies and solitary bees with average weight of 10-70 mg. The main pollinators of Ch. angustifolium and G. pratense are honey bees, bumblebees and wasps with average mass exceeding 70 mg.

The biochemical study of rat T-cell growth factor (interleukin 2).

T-cell growth factor (interleukin 2) was partially purified from the conditioned medium from Con A-stimulated rat splenocytes. It had the molecular weight of 23,000 according to gel filtration and in the range of 17,000-18,000 daltons according to PAGE in the presence of SDS. The molecular forms of IL 2 exhibited isoelectric points (pI) of 5.4-5.8 (major peak), 4.3-4.5 and 6.5-6.8 (minor peaks). T-cell growth-promoting activity was remarkably stable to temperature, pH, SDS and 2-ME treatments. Rat IL 2 did not bind to immobilized Con A, WGA, RC 1, and fucose-binding protein. The conditioned medium also contained the inhibitor of IL 2-dependent T-cell proliferation.

[Immunochemical determination of human lactoferrin]. / Immunokhimicheskoe opredelenie laktoferrina cheloveka.

The immunochemically pure preparation of lactoferrin was isolated from human colostrum and used for the immunization of animals with a view of obtaining antiserum, and also as a reference preparation for the determination of the content of lactoferrin in the standard. The monospecific antiserum to lactoferrin, obtained as the result of this procedure, was used for the determination of the content of lactoferrin in samples of human milk by the method of radial immunodiffusion. Through the content of lactoferrin in human milk showed considerable fluctuations, its level essentially decreased on the second week of lactation. In cases of the microbial contamination of milk the tendency towards an increase in the content of lactoferrin was observed irrespective of the time of lactation.

[Enzymatic activity of glycoprotein catabolism in the organs of rats with hyper- and hypovitaminosis A]. / Aktivnost' nekotorykh fermentov katabolizma glikoproteidov v organakh krys pri giper- i gipovitaminoze A.

Activities of glycosidases neuromanidase, alpha-L-fucosidase, beta-galactosidase and beta-N-acetylglucosaminidase were studied in rat small intestine and liver tissue under conditions of hyper- and hypovitaminosis A. Both excessive and insufficient administrations of vitamin A were accompanied by distinct alterations in activity of the enzymes studied in small intestine and (although less distinct) in liver tissue. The most significant dependence on the presence of vitamin A exhibited beta-galactosidase and especially alpha-L-fucosidase, activity of which was decreased in hypervitaminosis and increased in hypovitaminosis A. Activity of neuraminidase was usually slightly altered but its marked activation was noted in liver tissue under conditions of hypervitaminosis A. Vitamin A appears to participate in allosteric control of the glycosidase activity in small intestine and in catabolism of glycoproteins. The alterations of the enzymatic activity found in hyper- and hypovitaminosis A might be responsible for changes in composition of membrane glycoproteins and, hence, for the typical for vitamin A disbalance impairments in cellular growth and differentiation.