The nucleoid protein Dps binds genomic DNA of Escherichia coli in a non-random manner.

Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions accumulating them in the form of Fe2O3 within its protein cavity, interacts with DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis from some genes via competition with RNA polymerase or, vice versa, competing with inhibitors to activate transcription.

Development of a competitive double antibody lateral flow assay for the detection of antibodies specific to glycoprotein B of Aujeszky's disease virus in swine sera.

Three lateral flow assays (LFAs) for the detection of antibodies against glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera: a competitive double antibody sandwich LFA without a preincubation step (CDAS-gB-LFA), a CDAS-gB-LFA with a preincubation step (pCDAS-gB-LFA), and a competitive direct gB-LFA have been developed and were compared with each other and with a gB-ELISA. The assays are based on monoclonal antibodies to immunodominant epitopes of ADV gB. The pCDAS-gB-LFA proved to be the most specific and sensitive assay to detect antibodies directed to ADV gB. The specificity and sensitivity of the pCDAS-gB-LFA with the use of an LFA reader for test line intensity measurements were 97.6 and 94.9%, respectively. The lower diagnostic sensitivity of the pCDAS-gB-LFA compared to a gB-ELISA reflects its reduced analytical sensitivity, which was shown in titration experiments with positive sera. The pCDAS-gB-LFA, using the reader-based and visual detection modes, showed good agreement in respect to specificity; however, the LFA reader detection provided a higher diagnostic and analytical sensitivity compared to visual detection. The developed pCDAS-gB-LFA is a rapid, sensitive, and specific method for the detection of antibodies to ADV gB and can be used for screening ADV-infected swine in unvaccinated herds.

[The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90ß, we showed that membrane-bound Hsp90α and Hsp90ß play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90ß to the plasma membrane.

Effect of heat shock protein 90 (Hsp90) on migration and invasion of human cancer cells in vitro.

We studied the effect of purified native heat shock protein 90 (Hsp90) from bovine and mouse brain on migration and invasion of human glioblastoma (A-172) and fibrosarcoma (HT1080) cells. Hsp90 in concentrations of 0.01-0.10 mg/ml stimulated migration and invasion of tumor cells in vitro by 20-32% (p<0.05). Polyclonal antibodies to Hsp90 blocked the Hsp90-dependent stimulation of cell invasion, which indicates specificity of the stimulating effect of extracellular Hsp90 on tumor cell invasion. Hence, extracellular Hsp90 can be considered as a promising molecular target, because its inhibition can suppress invasion and metastasizing of tumor cells.

[Immunological characteristics of Aujeszky's disease virus glycoprotein].

A panel of monoclonal antibodies (MAbs) specific to glycoprotein D (gD) of Aujeszky's disease virus (ADV, Suid herpesvirus 1) was produced and characterized. MAbs were used to identify 9 topologically different epitopes and epitopic groups on gD. The majority of the identified epitopes were conformational. Most gD-specific MAbs possessed virus-neutralizing activity in the presence and absence of the complement. MAbs neutralized the virus at the stage of its penetration into the cell and inhibited the cell-to-cell spread of viruses. Two immunodominant epitopes and one immunodominant domain that induce the most prominent humoral immune response were identified when the animals were infected and immunized. A method was developed for affinity purification of ADV glycoprotein D. Immunization of mice with affinity-purified gD induced a strong humoral immune response and protected mice against lethal ADV challenge. In passive immunization, the majority of gD-specific MAbs protected mice against infection. The findings confirm the important role of ADV glycoprotein D in inducing protective anti-ADV immunity.

[Interaction of the glycoprotein gB of Aujeszky's disease virus (Suid herpesvirus 1) with cellular heparan sulfates].

The interaction of the glycoprotein gB of Aujesky's disease virus (ADV, Suid herpesvirus 1) with heparan sulfates of BHK-21 cells was studied. The study used the native glycoprotein gB purified from the virus envelope by affinity chromatography based on gB-specific monoclonal antibodies. The cellular binding of the glycoprotein gB was specific and dose-dependent. Heparin, a structural analogue of heparan sulfates, inhibited the cellular binding of gB. The herapinase treatment of cells also resulted in the inhibition of the cellular binding of gB. The purified glycoprotein gB bound to heparin-Sepharose and was specifically eluated by heparin. The denaturation of gB was followed by 70-80% decreases in its binding to Sepharose-immobilized heparin. The findings may lead to the conclusion that plasma cell membranous heparan sulfates are one of the glycoprotein gB receptors in ADV. At the same time the heparin-binding site of ADV gB is at least partially conformation-dependent.

[Incorporation of glycoproteins of the Aujeszky's disease virus ( Suid herpesvirus 1) into artificial liposome membranes and their interaction with cells]. / Vstraivanie glikoproteinov virusa bolezni Aueski (Suid herpesvirus 1) v membrany iskusstvennykh liposom i ikh vzaimodeistvie s kletkami.

The purpose of the case study was to investigate the interplay between liposomes, containing the in-built glycoproteins of the Aujeszky disease virus (ADV, Suid herpesvirus 1) with plasmatic membranes of sensitive cells. The conditions of reconstructing the ADV glycoproteins into artificial-liposome membranes were optimized. The above liposomes (virosomes), 40 x 200 nm, were impermeable to univalent ions, which confirmed the virosome membranes were intact. The gE and gB glycoproteins (90-98% of them) were located, inside the liposome membrane with the outwards orientation of their ecto-domain fragments. Virosomes were binding with cells in the dose-dependent mode. The purified ADV virions, the ADV gB glycoproteins and heparin inhibited such binding process of virosomes with cells, which denoted the specificity of their interaction with cells. An effective internalization of cell-binding virosomes was observed at 37 degrees C. The conclusion is that the ADV glycoproteins, constructed into the liposome membranes, simulate adequately enough the viral receptor structures and that the thus obtained virosomes could be used to investigate the interplay between alpha-herpes viruses and cells.

[Immunological and functional characteristics of epitopes and regions of gB glycoprotein of Aujeszky's disease virus]. / Immunologicheskaia i funktsional'naia kharakteristika épitopov i oblastei glikoproteina gB virusa bolezni Aueski.

The role of different gB epitopes and regions at some stages of virus replication in cell cultures and in the formation of immunity to Aujeszky's disease virus (ADV) was studied using a panel of 13 monoclonal antibodies (MAB) that recognize glycoprotein gB (gB) of ADV and antisera against fusion recombinant proteins expressing gB fragments. Productive infection following virion attachment was prevented by antibodies to the N-terminal domain of gB. Three MABs against the N-terminal domain of gB and 5 MABs directed against the immunodominant region located in the gBc-subunit of gB inhibited the cell-to-cell spread of viral infection. After immunization with recombinant proteins expressing the N-terminal fragments of gB 80% mice were protected from lethal ADV challenge. After passive immunization the majority of MABs protected 20-80% mice from lethal ADV challenge. Hence, the N-terminal domain of ADV gB is associated with the virus penetration into the cell and is important for anti-ADV immunity.

[Effect of centimeter microwaves on the antibody production in mice]. / Vliianie nizkointensivnykh élektromagnitnykh voln santimetrovogo diapazona na uroven' antiteloobrazovaniia u myshei.

The effect of low-intensity microwaves (8.15-18 GHz, 0.3 or 1 microW/cm2, 1.5 h daily for 30 days) on antibody production in healthy male NMRI mice after immunization with affinity-purified carboanhydrase isolated from bovine erythrocytes with and without Freund's adjuvant was studied. It was found that exposure to microwaves leads to an increase in the concentration of antibodies in blood plasma, the stimulating effect being more pronounced in the primary immune response. It is assumed that the effect of enhancement of the immune response by the action of centimeter microwaves can be used in the adjuvant therapy.

[Development of immunoenzyme methods for detecting antibodies to Aujeszky's disease virus gB glycoprotein in swine serum]. / Razrabotka immunfermentnykh metodov vyiavleniia antitel k glikoproteinugB virusa bolezni Aueski v syvorotke svinei.

Four ELISA methods have been developed for detecting antibodies to Aujeszky's disease virus (ADV) glycoprotein gB. Indirect ELISA is based on affinity-purified gB (affi-gB-ELISA); three blocking ELISAs: indirect blocking ELISA (lbgB-ELISA), direct blocking ELISA (db-gB-ELISA), and two-site "sandwich" ELISA (sb-gB-ELISA) are based on monoclonal antibodies to conservative immunodominant epitopes of gB. The specificities and sensitivities of ELISAs were compared with each other and with indirect ELISA based on purified ADV virions (vir-ELISA). Affi-gB ELISA, db-gB-ELISA, and sb-gB-ELISA possess 100% sensitivity, ib-gB-ELISA 98% sensitivity, and vir-ELISA 93% sensitivity. Affi-gB ELISA, ib-gB-ELISA, db-gB-ELISA, and sb-gB-ELISA possess 100% specificity and vir-ELISA 92% specificity. The efficiency of detection of ADV-specific antibodies by affi-gB ELISA, db-gB-ELISA, and sb-gB-ELISA was comparable to that of analogous commercial test. Since db-gB-ELISA is easier to perform than affi-gB-ELISA or sb-gB-ELISA, it is concluded to be the most appropriate test for detecting pigs infected with ADV among non-vaccinated animals.

Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals.

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.

Distribution of B-cell epitopes on the pseudorabies virus glycoprotein B.

In order to map antigenically important regions of glycoprotein B (gB) of pseudorabies virus (PrV), a panel of recombinant fragments of gB expressed in E. coli and truncated fragments of gB generated by cleavage of purified native gB with trypsin and cyanogen bromide was analysed by using 26 monoclonal antibodies directed against gB. Three continuous epitopes were localized in the vicinity of the N terminus of gB, between amino acids (aa) 59 and 126. One continuous epitope mapped between residues 214 and 279. The residues involved in the assembly of eight discontinuous epitopes were located between aa 540 and 734. The constituents of two discontinuous epitopes were harboured in a segment encompassing aa 540-646. The clustering of continuous epitopes at the extreme N terminus of PrV gB and the locations of residues involved in the assembly of discontinuous epitopes of PrV gB are in good agreement with data on epitope locations in gB homologues from other herpesviruses.

Isolation of mutants of Aujeszky's disease virus with antigenically altered glycoprotein E by affinity chromatography using monoclonal antibodies.

An efficient method for isolation of virus mutants with antigenically altered proteins is described. The method is based on the separation of viruses with wild-type and antigenically altered proteins by affinity chromatography using monoclonal antibodies (MAbs). A nonessential glycoprotein E (gE) of Aujeszky's disease virus (ADV) was chosen as a model for introducing the antigenic changes. The ADV strain Ka mutagenised with 5-bromo-2'-deoxyuridine was used for the selection of mutants that do not bind to gE-specific MAb conjugated to resin. After three rounds of isolation by affinity chromatography, the resulting viruses that escape the binding to MAb were plaque-purified by plating at limiting dilution, and virus isolates were tested by the gE-specific sandwich ELISA in which the selecting MAb was used as a capture antibody. About 70% of the ADV isolates tested were not recognised by the sandwich gE-ELISA. The analysis of some of virus isolates in indirect ELISA with a panel of 16 gE-specific MAbs revealed that at least several of the generated virus isolates were mutants expressing gE with alterations in the epitope of the selecting MAb 75/7, as well as in the majority of other conformation-dependent epitopes of gE. The method for the production of antigenically altered viruses by affinity chromatography using MAbs is simple and convenient, and can be utilised with MAbs irrespective of their virus-neutralising activity.

Glycoprotein B of Aujeszky's disease virus: topographical epitope mapping and epitope-specific antibody response.

A panel of 26 monoclonal antibodies (mAbs) against glycoprotein B (gB) of Aujeszky's disease (pseudorabies) virus (ADV), a glycoprotein complex consisting of three glycoproteins, gBa, gBb, and gBc, was produced by two research groups and was used for the topographical epitope mapping of gB. An epitope map was constructed in which the identified epitopes of gB were situated in 14 topologically distinct antigenic domains; ten antigenic domains represented by 22 mAbs were localized on gBc, while four antigenic domains represented by four mAbs resided on gBb of the gB complex. All the epitopes located on gBc appeared to be conformation-dependent, whereas all the epitopes on gBb were conformation-independent. The identified epitopes of gB were conserved among laboratory, vaccine and field ADV strains. Conformation-dependent epitopes were shown to contribute largely to the overall antibody response to gB in naturally infected swine and immunized mice. Moreover, it was found that most of the infected animals responded relatively weakly to the identified conformation-independent epitopes of gB, while a group of immunodominant epitopes that induced a strong antibody response was represented exclusively by conformation-dependent epitopes from different antigenic domains. The results clearly demonstrated that conformation-dependent epitopes of gBc play a crucial role in inducing the humoral immune response to gB of ADV during the natural infection of swine and immunization of mice. The application of mAbs of our panel as research and diagnostic tools is discussed.

Immunological characterisation of glycoprotein E of Aujeszky's disease virus.

A panel of 14 monoclonal antibodies (MAbs) against glycoprotein E (gE) of Aujeszky's disease (pseudorabies) virus (ADV), which constitutes a representative sample of naturally occurring gE-specific antibodies in sera from infected animals, was produced and characterised. Eleven topologically distinct antigenic domains represented by one or more MAbs were identified on gE by using these MAbs and three additional gE-specific MAbs. Three of the MAbs available recognised conformation-independent epitopes on gE, while the other 14 MAbs bound to conformation-dependent epitopes. By using the recombinant protein encompassing the N-terminal part of gE, which was expressed in Escherichia coli, all the conformation-independent epitopes of gE were mapped within the first 125 amino-terminal amino acids of gE. The epitopes of gE were demonstrated to be conserved among gE-positive laboratory, field and vaccine ADV strains. Conformation-dependent epitopes were shown to contribute largely to the overall antibody response to gE in naturally infected swine and immunised mice. Most of the infected animals responded weakly to the identified conformation-independent epitopes of gE, while the group of immunodominant epitopes of gE was represented exclusively by conformation-dependent antigenic determinants from different antigenic domains. The results clearly demonstrated that conformation-dependent epitopes play a crucial role in inducing the humoral immune response to gE of ADV during the natural infection of swine and immunisation of mice. The application of MAbs of our panel as research and diagnostic tools is discussed.

Glycoprotein gE blocking ELISAs to differentiate between Aujeszky's disease-vaccinated and infected animals.

Three blocking ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera (an indirect blocking ELISA (gE-iELISA), a direct blocking ELISA (gE-dELISA) and a two-site 'sandwich' blocking ELISA (gE-sELISA)) have been developed. The gE-ELISAs are based on monoclonal antibodies (mAbs) directed to gE and detect gE-specific antibodies in sera by blocking the binding of mAbs to one (in the gE-iELISA and the gE-dELISA) or two (in the gE-sELISA) epitopes of gE. From a panel of thirteen gE-specific mAbs, mAbs, their conjugates and the combination coating mAb/conjugate that optimally detect anti-gE antibodies in the assays were selected. Sera from uninfected unvaccinated swine or swine vaccinated against different swine viral disorders were negative by three gE-ELISAs, the blocking gB-ELISA, and VNT. Swine vaccinated with gE-negative vaccine were seropositive in the gB-ELISA and VNT but were seronegative by three gE-ELISAs. Infected unvaccinated swine, infected swine vaccinated with gE-negative vaccine, and swine vaccinated with gE-positive vaccine were detected as seropositive by three gE-ELISAs as well as in the gB-ELISA. The gE-dELISA and the gE-sELISA proved to be specific and the most sensitive and reliable assays to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine. These two gE-ELISAs were at least as sensitive as the gB-ELISA for detecting ADV-specific antibodies.

[Monoclonal antibodies to 8-oxo-2'-deoxyguanosine (8-hydroxyguanosine). Characteristics and use for determining DNA damage by active forms of oxygen]. / Monoklonal'nye antitela k 8-okso-2'-dezoksiguanozinu (8-gidroksiguanozinu). Kharakteristika i ispol'zovanie dlia opredeleniia povrezhdeniia DNK aktivnymi formami kisloroda.

It has presently been established that the guanine base is one of the most sensitive and biologically significant target for the damaging action of active oxygen species on DNA, 7.8-dihydroxy-8-oxoguanine (8-oxoguanine, 8-hydroxyguanine) being the major degradation product and the most essential biomarker for DNA damage by active oxygen species. Murine monoclonal antibodies (MAbs) specifically recognizing 8-oxoguanine have been raised. Using competitive solid phase immunoenzymatic assay. (IEA) with peroxidase-antiperoxidase (PAP-method), a quantitative assay of this degradation product and characterization of affinity and specificity of MAbs have been carried out. The affinity constant (Kaff) Mabs for 8-oxo-2'-deoxyguanosine is equal to 1.3-10(6), that for 8-oxo-guanosine-to 1.10(6), exceeding by more than three orders of magnitude the Ka values for natural guanyl nucleosides and other possible cross-structure analogs. IEA was used to determine the DNA degradation product in gamma-irradiated DNA. The radiation-chemical yield of 8-oxoguanine (G = 0.57 molecules per 100 eV) is consistent with those obtained by other methods.

[Use of monoclonal antibodies for detecting Aujeszky's disease virus in culture media by an immunoenzyme method]. / Ispol'zovanie monoklonal'nykh antitel dlia opredeleniia immunofermentnym metodom virusa bolezni aueski v kul'tural'noi srede.

Three modifications of quantitative enzyme immunoassay (EIA) for assessment of Aujeszky's disease virus (ADV) in culture medium are compared, in which monoclonal antibodies (MAB) to ADV GH glycoprotein were used: nonconcurrent two-site "sandwich" EIA on the basis of two different MABs, indirect concurrent EIA, and direct concurrent EIA. MABs fit best of all for use in every of EIA modifications were selected. Conditions of the assays were optimized. Concurrent EIAs were 10-20 times less sensitive than sandwich EIA. The developed sandwich EIA permitted ADV assay in preparations of both live and formaldehyde-inactivated virus.

[The isolation and characteristics of monoclonal antibodies to the glycoprotein GII of Aujeszky's disease virus and their use for the epitopic mapping of GII]. / Poluchenie i kharakteristika monoklonal'nykh antitel k glikoproteinu GII virusa bolezni Aueski i ikh ispol'zovanie dlia épitopnogo kartirovaniia GII.

A panel of 24 different monoclonal antibodies (MAB) to Aujeszky's disease virus (ADV) proteins was prepared. Seven MABs were directed to ADV glycoprotein GII (6 MABs to GIIc chain and 1 MAB to GIIb chain). GII epitope mapping with the resultant MABs was carried out. Four ADV GII epitopes were detected: epitopes A, B, and C located on GIIc chain and epitope D located on GIIb chain. Epitope B overlaps epitopes A and C which do not overlap each other, epitope D does not overlap other epitopes. Epitopes A, B, and C are located in a restricted area of GIIc chain which seems to be one of GII immunodominant regions. The detected epitopes are mainly conformational, for they are sensitive to denaturation. Of all MABs obtained more than 30% were directed to ADV GII. This indicates that GII is one of the most important ADV antigens. All the seven MABs to GII interacted with all tested ADV strains (K. Arsky, VGNKI, BUK, and NIA-4), this being in line with the data on a highly conservative nature of ADV GII.