[Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation.

CD150-mediated Akt signalling pathway in normal and malignant B cells.

AIM: To study upstream and downstream events in CD150-mediated Akt signaling pathway in normal human B cells, EBV-transformed lymphoblastoid (LCL) and malignant Hodgkin's lymphoma (HL) B cell lines. METHODS: To access protein-protein interaction we applied immunoprecipitation, Western blot analysis and surface plasmon resonance (SPR) technique. A novel modification of SPR technique using reduced glutathione bound to golden surface was proposed. Immunostaining and isolation of cytoplasmic fractions and nuclear extracts were performed to detect proteins' localization in cells. Western blot analysis was performed to follow up the phosphorylation of proteins on specific sites and proteins' expression level. RESULTS: It was shown that CD150 ligation induced Akt activation in normal tonsillar B cells (TBC), SH2D1A positive LCL and HL B cell lines. The p85α subunit of PI3K co-precipitated with CD150 cytoplasmic tail. This direct association depends on tyrosine phosphorylation and is mediated by N terminal SH2 domain of p85α. CD150 initiated phosphorylation of FoxO1 transcription factor in normal B cells as well as in LCL MP-1 and HL cell line L1236. At the same time, CD150 ligation triggered GSK-3ß kinase phosphorylation only in immortalized LCL MP-1 and HL cell line L1236. CONCLUSIONS: We have demonstrated that CD150 receptor could trigger PI3K-mediated Akt signaling pathway in normal, EBV-transformed and malignant B cells. CD150-mediated phosphorylation of Akt downstream targets GSK-3ß and FoxO1 in EBV-transformed and HL cells could be one of the mechanisms to avoid apoptosis and support survival program in these immortalized B cells.

[Immobilization of the glutathione by the complementary coordination binding].

A simple method for immobilization of biologically imporant molecules with many functional fragments by selective binding of their thiogroups with the surface caroxyl groups by cadmium ions was proposed. Biofunctional properties of these structures were studied by surface plasmon resonance method on the model of the glutathione (GSH), which was immobilized by means of mixed (a:b form 1:100 o 1:700) thiol monolayers with terminal groups of the methyl/hydroxyl (b) and carboxyl (a) type. The maintenance of the biofunctional conformation ofglutathione-S-transferase (GST) after its interaction with GSH was checked by the use of specific anti-GST antibodies. It was shown that CH3 matrix has considerable non-specific binding and is not suitable for the formation of the biofunctional GST layer. At the same time OH-based structures demonstrate specific interaction GST-anti-GST, the stoichiometry of which corresponds to the bidentate binding. Considered simple method of the immobilization can be used to create the functional surface architectures in the analyticasl biochemistry and chemical analysis.

Kinetic studies of protein-surface interactions: A two-stage model of surface-induced protein transitions in adsorbed biofilms.

The irreversible adsorption of proteins on artificial surfaces plays an important role in a wide variety of practical problems. The simple analytical models based on definite concepts regarding the mechanisms of interfacial evolution can be used efficiently for characterization of protein-surface interactions by analyzing the intrinsic kinetics of the process. In this article, analytical expressions are derived for the adsorption kinetics that take into account the presence of more than one adsorbed state for proteins in biofilms. It is shown that the experimentally observed dependence of the adsorbed mass on the concentration of protein in solution can be reproduced with this model, and the approach provides a rapid method for obtaining quantitative parameters for the adsorption process. It is shown by analytical approximation of the kinetic curves for fibrinogen adsorption onto an unmodified gold surface studied by a surface plasmon resonance biosensor that this model is in good quantitative agreement with experiments. It is found that the rate of adsorption, controlled mainly by the mass flow from the solution, determines the contribution both to self-assembling and spreading, resulting in variations of adsorbed fibrinogen interfacial structures.

[Detection of antigen-antibody interaction of human adenovirus by the method of surface plasmon resonance].

A possibility to detect adenoviral protein--hexon, using specific antibodies by surface plasmon resonance (SPR) was demonstrated. The hexon of the human adenovirus 2 (Ad2) binds to antibodies immobilized on the sensor surface treated by KNCS and protein A Staphylococcus aureus. The specificity of antihexon antibodies was demonstrated by indirect method of fluorescent antibodies (MFA) and cellular variant of the immunoassay (cELISA).

[Immunospecific detection of the potato virus X by the surface plasmon resonance].

Highly specific and proximal method of laboratory diagnostics of the viral disease, has been developed using the structural protein of the potato virus X, as a model, and monospecific antibodies to it. The immunospecific determination of the potato virus X was carried out by the surface plasmon resonance method using specific IgG-antigen complexes, immobilized on the sensor surface modified by rodanide and protein A of Staphylococcus aureus.

Detection of plant viruses using a surface plasmon resonance via complexing with specific antibodies.

The use of instrumental systems based on the surface plasmon resonance (SPR) for rapid diagnosis of intact plant viruses (in particular, tobacco mosaic virus (TMV)) is considered. A new approach using detection of viral antigen and antibody (IgG) complexes formed during the preincubation step (instead of their consecutive application in classical approach) is discussed. A comparison between signal level registered from the mixture of virus and specific serum and that from the sample without virus (samples deposited onto the sensor surface treated with thiocyanate and protein A Staphylococcus aureus) allows unambiguous detection of viral particles in the material studied. The performance capabilities of the method are discussed and illustrated by quantitative detection of virus in the actual samples (cells homogenate) at high concentration.

[Investigation of interaction of immunoglobulins and detection of viral antigens in cell homogenates by the surface plasmon resonance method]. / Doslidzhennia vzaiemodiï imunohlobuliniv ta vyiavlennia virusnykh antyheniv u klitynnykh homohenatakh metodom poverkhnevoho plazmonnoho rezonansu.

Immobilization of immunoglobulins on the unmodified gold surface and the gold surface, modified by thiocyanate and protein A Staphylococcus aureus and their subsequent interaction with complementary antibodies were studied using the surface plasmon resonance method. Viral antigens were detected by surface plasmon resonance method in the cell homogenate of green alga Bracteococcus minor, which was artificially infected by tobacco mosaic virus. It was shown, that the process of interaction between virus and antiviral serum obey the Langmure model.

[Method of orienting immobilized proteins on gold surface sensory modified with rhodanide]. / Sposob orientirovannoi immobilizatsii belkov na modifitsirovannykh rodanidom zolotykh poverkhnostiakh sensora.

The behavior of the biomolecules near the charged surface depending on the pH level of the solution was studied by the method of surface plasmon resonance. It was shown, that the preliminary modification of the surface by thiocyanat generating the effective negative charge at the surface allowed to immobilize protein molecule orientation depending on their charge state. Proteins keep their biological properties and form the oriented monolayer in the wide range of pH.

Simple method for plant virus detection: effect of antibody immobilization technique.

The possibility has been demonstrated for applying a surface plasmon resonance for detecting plant viruses in real samples. An optimal mode for antiviral immunoglobulin immobilization on sensor surfaces is described. Out of three proposed techniques for sensor surface treatment, namely, unmodified gold surface, gold surface treated with (a) thiocyanate and (b) thiocyanate and protein A (Staphylococcus aureus), the latter was chosen as most suited for retention of the formed native immunoglobulin layer.

[Orientation of proteins on the sensor surface and optimization of their immobilization by prior protection of the active center]. / Orientatsiia proteinov na poverkhnosti sensora i optimizatsiia ikh immobilizatsii putem predvaritel'noi zashchity aktivnogo tsentra.

When studying biospecific interactions with application of surface plasmon resonance, one of the main problems is reagent proper orientation to the sensor surface. Due to rather high chemical activity of molecular receptor sites, the interaction between these areas and surface may become predominant. Here we propose a technique for prevention of such orientation of bioreceptors using soybean trypsin inhibitor STI as an example. To obtain oriented STI immobilization on a modified gold surface its active site has been previously blocked through interaction with its specific partner trypsin. After conjugate immobilization on the sensor surface the components were separated using a glycine buffer (pH 2.2).

Analysis of the response of planar polarization interferometer to molecular layer formation: fibrinogen adsorption on silicon nitride surface.

The most sensitive optical method of interferometry was exploited for determination of changes in the refractive index following the adsorption of biological molecules onto the solid surface. Instead of having two waveguiding arms (the main and the reference) in traditional Mach-Zhender interferometer, two ortogonal TM and TE modes propagating through the SiO(2)-Si(3)N(4)-SiO(2) waveguide structure were employed in planar polarization interferometer (PPI). Multiperiodic PPI response was, therefore, formed due to the phase shift between TM and TE modes. A matrix simulation procedure was developed in order to investigate the influence of both the refractive index and molecular layer thickness on the PPI response. Nonspecifical binding of fibrinogen to silicon nitride surface was studied as a model object for PPI testing. The results obtained are in good agreement with the known information about fibrinogen adsorption on the different surfaces. An attempt to introduce the concept of 'surface molecular concentration and molecular polariziability' instead of 'molecular layer thickness and refractivity' was undertaken.