[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.

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 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.

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.