Receptor of fibronectin edb-domen

FIELD: biotechnology, medicine.

SUBSTANCE: cells are contacted with protein containing fibronectin EDb-domen in presence of one or more chemical substances. As check experiment reaction between the same cells and abovementioned protein in absence of said substances. Expression of certain protein or presence of nucleic acid encoding the same makes it possible to select compounds bonding to fibronectin EDb-domen.

EFFECT: new biotechnological method.

1 tbl, 3 ex

 

The present invention relates to a protein (protein)that specifically bind to the EDb-a domain of fibronectin.

Fibronectin represent an important class of matrix glycoproteins. Their main role is to ensure the adhesion of cells to many different extracellular matrices. The fact that fibronectin are present on the surface of normal cells in culture and not in transformed cells, allowed us to identify fibronectin as important adhesion proteins. Fibronectin interact with a large number of various other molecules, such as collagen, heparinotherapie and fibrin, and thereby regulate cell shape and formation of the cytoskeleton. In addition, fibronectin responsible for the migration and differentiation of cells during embryogenesis. In addition, fibronectin play an important role in the healing process, providing the movement of macrophages and other immune cells in the affected area, as well as in the formation of blood clots, providing adhesion of platelets to the damaged areas of blood vessels.

Fibronectin are dimers of two similar peptides, the length of each chain is approximately 60-70 nm. Currently identified at least 20 different what's fibronectine circuits, all of which can be obtained by alternative splicing of the RNA transcript of a single gene of fibronectin. Analysis of the proteolytic cleavage of fibronectin has allowed to establish that the polypeptides consist of six characterised by a high degree of folding of domains, each of which in turn contains the so-called re-sequence ("repeats"), which is based on the similarity of their amino acid sequences can be classified into three types (type I, II, III). The Central region of both chains of the dimer is a plot consisting of the so-called type III repeats, which contain an average of 90 amino acids (A.R. Kornblihtt, Vibe-Pedersen, K., and F.E. Baralle, Isolation and characterization of cDNA clones for human and bovine fibronectins, Proc. Natl. Acad. Sci. USA 80 (1983), cc.3218-3222). According to the results of structural studies, it was found that each repeat type III consists of seven beta-chains, arranged in two parallel planes, with a short loop region represent potential sites of interaction protein-protein (D.J. Leahy, W.A. Hendrickson, Aukhil I. and Erickson, H.P. Structure of fibronectin type III domain from tenascin phased by MAD analysis of the selenomethionyl protein. Science, 258 (1992), cc.987-991). These repetitions of type III fibronectin function as adhesion molecules that interact with those present on the cell surface molecule and, the so-called "integrins". The term "integrin" was first used in 1987 in a review article (R.O. Hynes, Cell 48 (1987), cc.549-550) to denote a group of related heterodimeric present on the cell surface molecules that function as intermediaries between the extracellular matrix and the intracellular cytoskeleton and thus induce adhesion and migration of cells. Thus, such heterodimeric receptors "integrate" or transmit signals from the surrounding cell from the external environment, exerting specific effects on the cell. Currently known 17 beta-subunits that are capable of specifically and ecovalence to interact with more than 20 alpha-subunits, thus forming more than 20 different families (Plow E.F., etc., J. Biol. Chem., 275 (2000), cc.21785-21788). Interaction of fibronectin with at least 8 different integrins is mediated primarily by the RGDS sequence present in the tenth type III repeat of fibronectin (III-10). In addition, it was found that at least four integrin can interact with fibronectin-independent RGDS way (Plow E.F., etc., J. Biol. Chem., 275 (2000), cc.21785-21788). The group repeated sequences of type III in addition to III7-, III8-, III9 - III10 sequences are also repetitions EIIIB and EIIIA (EDband EDa). The functions of both of these re the latter is of valnontey so far not found or revealed only to a small extent. The results of research conducted Jarnagin W. and others (Jarnagin W., Rockey D., V. Koteliansky, S. Wang and D. Bissell, Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis, J. Cell Biol., 127 (1994), cc.2037-2048), suggest that the EDa-domain is involved in the early response of the liver to injury and in addition EDadomain probably involved in mediating the processes of cell adhesion. Although the isoform of fibronectin, which contains the sequence of the EDb(EDb-FN ED-B or EDB), and is undetectable in healthy tissue of adult individuals, however it shows strong expression in fetal tissues and in tumor tissue, as well as in the healing process.

In the process of tumor development occurs due to proteolytic degradation of the existing components of the matrix rebuilding of extracellular matrix tissue in which the tumor growth. This forms induced by the tumor extracellular matrix, which is different from the matrix of healthy tissues, creates more favorable for the growth of the tumor environment and promotes angiogenesis. Angiogenesis is one of the most important processes during tumor growth, and this is defined as the process by which existing covered by vascular endothelial formation of new blood vessels. Angiogenesis p is ecstasy an invasive process, the necessary conditions for the flow are the proteolysis of the extracellular matrix, proliferation, directed migration, and differentiation of endothelial cells with the formation of new capillaries, promote the growth of tumors beyond a certain size.

Currently, a correlation was found between the EDb-a domain of fibronectin (EDb-FN) and tumor growth. In addition, the EDb-FN accumulated in the process of angiogenesis new blood vessels, thereby is indicative of the sign of angiogenesis (Castellani p, Viale G., Dorcaratto A., Nicolo G., Kaczmarek J., Querze g, Zardi L. Int. J. Cancer, 59 (1994), cc.612-618).

EDb-domain is composed of 91 amino acid repeated sequence of type III, which has an extremely high degree of homology with sequences of fibronectin rats and chickens component from 96 to 100%. In this domain there are no RGDS sequence or other amino acid sequence for which it is known that they mediate the interaction with integrins. The exact function of the ED-B domain to the present time has not been settled. In three published on this subject in the works, listed below, were made assumptions about the total incentive function on the adhesion/proliferation of various cell types.

In the work of Chen and Culp, publish vannoy in Exp. Cells Res. 223 (1996), cc.9-19, says that the cellular fibronectin contain EDbdomain and related repeated sequences of type III as stimulating, under certain conditions, the adhesion sequences, which can be adjusted using cells by alternative splicing of the primary transcript of fibronectin. According to the results of more recent studies (Chen and Culp, Clin. Exp. Metast., 16, 1 (1998), cc.30-42) found that EDbdomain induces the activation of cells, which leads to tyrosine phosphorylation of focal adhesion proteins, namely the mechanism that is different from the mechanism, mediated by repeated sequences of type III8-9-10, recognizing integrins. At the same time among a growing number of experts, most agree that the adhesion of cells to extracellular matrix, respectively, to other cells, is an important source cell activation and thereby responsible for regulation of many processes, such as growth, differentiation and transformation of cells. Induced adhesion signal is the activation of protein-tyrosinekinase and includes a cascade of tyrosine phosphorylation of various signaling molecules. The authors of the above studies point to the fact that in this process of signal transmission plays a major role focal edges the district kinase (FAK) with a molecular mass of 125 kDa, which allows you to set the relationship between the interaction of cells with extracellular matrix proteins and the activation of intracellular signaling molecules such as Src (Xing Z., Chen H.C., Nowlen J.K., S.J. Taylor, D. Shalloway, and J.L. Guan, Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain, Mol. Biol. Cell, 5 (1994), cc.413-21), Grb2 (D.D. Schlaepfer, Hanks S.K., Hunter T. and van der Geer p, Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase, Nature, 372 (1994), cc.786-791) and PI-3-kinase (H.C. Chen and J.L. Guan, Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase, Proc, Natl. Acad. Sci. USA, 91 (1994), cc.10148-10152). Another focal adhesion protein p130cas also assumes that it is involved in mediated adhesion signaling and specific carcinogenic activity, although its exact function is currently full and not detected (Sakai R., Iwamatsu, A., Harada N., and others, A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and c-Src in a tyrosine phosphorylation-dependent manner, EMBO J. 13 (1994), cc.3748-3756; Petch L.A., Bockholt S.M., Bouton, A., J.T. Parsons, and K. Burridge, Adhesion-induced tyrosine phosphorylation of the p130 SRC substrate, J. Cell. Sci., 108 (1995), cc.1371-1379; T.R. Polte and Hanks S.K., Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p 130Cas, Proc. Natl. Acad. Sci USA, 92 (1995), cc.10678-10682).

According to research conducted by Chen and Culp (1998, see above), it was found that having one repeat protein EDbstimulated the proliferation of cells of Balb/c 3T3, and phosphorylation of tyrosine with the participation FACTORS more intensively than adjacent repetitions III8, and so what. It has been suggested that at physiological concentrations of cellular fibronectin binding tetrapeptide RGDS, part III 10, integrins likely induces intense enough for cell adhesion signal, and therefore the mechanism, mediated by interaction III10 with integrin, does not lead to reaction of tyrosine phosphorylation. Next, it is assumed that the difference in reactions to different ways mediated by various factors cell adhesion is due to the different activation of a variety of small GTP-binding proteins. Three of these proteins (cdc42, rac and rho), members of the ras superfamily, plays an important role in the morphological changes of cells. Gene cdc42 is located in the sequence against the course of transcription relative to the rac and directly induces the formation of filopodia (C.D. Nobes and Hall, A., Rho, Gus, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia, Cell 81 (1995), SS-62). In this case, the activation of rac is responsible for the formation of lamellipodia and the network of actin filaments between filopodia. Below in the course of transcription of the gene rho can be activated under the influence of rac and induces focal adhesion and the formation of actin filaments. All these phenomena depend on the activation of tyrosine kinase, and in FACT is expected to participate in the t in these processes. In the work of Chen and Culp suggested that morphological differences between cells, which have adhesion due to 7-EDb-8, and cells that have adhesion due 8-9-10, based on different activation of the small GTP-binding proteins. From this it follows that the adhesion caused 8-9-10, using mediated by integrin transmission signal ultimately leads to activation of rho, causing focal adhesion and the formation of actin filaments, whereas the adhesion of cells of Balb/c 3T3 conditioned 7-EDb-8, only leads to the activation of cdc42 and rac proteins, but not to activation of the rho gene. However, none of these two works is not the data, confirming the above assumptions.

According to the results of another study (Hashimoto-Uoshima etc., J. Cell Sci. 110 (1997), s-2280), it was found that cell adhesion of cultured fibroblasts is enhanced in the presence of fragments of fibronectin, including EDb-a domain of fibronectin. From this it follows that subjected to splicing EDbdomain may have an important biological function concerning the enhancement of adhesion and proliferation of cells. In contrast, the inclusion of the EDadomain in these fragments in the absence of the EDbdomain prevents normal focal adhesion cells. Based on this, the authors of the decree of the frame of the study conclude that the inclusion of both domains in the molecule fibronectin may be a mechanism whereby the adhesion of the cells reaches a level that facilitates the process of their movement, for which the necessary adhesion and termination of cell adhesion.

Research conducted on chicken embryos and adult mice were allowed to establish that mediated by the EDbdomain angiogenesis can be blocked by inhibition of integrin α3β1 endothelial cells (Renato and others, AACR, LB-60 (2001)).

None of the above studies is not given a clear answer about the functions of the EDbdomain and is not given any information about the identity of the potential(s) receptor(s) EDbdomain.

Based on the foregoing, the present invention was used to further identify the functions of the EDb-domain. Another object of the invention consisted in the identification of specific receptor EDb-domain. The next task of the invention was to identify the specific characteristic of the EDbmechanism of adhesion and interaction with receptor molecules that can participate in the process of angiogenesis. Another object of the invention consisted in identifying areas EDbresponsible for specific binding.

This task is solved with the help of protein,

a) which has the ability to specifically bind with the EDb-a domain of fibronectin,

b) which is specifically expressed or activated in endothelial cells,

in) which is specifically expressed or activated in stromal cells of the tumor,

g) which is specifically expressed or activated in tumor cells,

d) linking with EDbdomain amenable to inhibition by using the polypeptide and

(e) the apparent molecular weight determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS page-LTO-electrophoresis), is from 120 to 130 kDa for the light chain and 150 to 160 kDa heavy chain.

Preferred is primarily protein,

a) which has the ability to specifically bind with the EDb-a domain of fibronectin, and the binding region is characterized by the presence of at least one sequence selected from the group comprising SEQ ID NO: 1-3,

b) which is specifically expressed or activated in endothelial cells,

in) which is specifically expressed or activated in stromal cells of the tumor,

g) which is specifically expressed or activated in tumor cells,

d) linking with EDbdomain fibronectine inhibition using the polypeptide, which contains a sequence selected from the group comprising SEQ ID nos: 1-3, and

(e) the apparent molecular weight determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate ranges from 120 to 130 kDa for the light chain and 150 to 160 kDa heavy chain.

Most preferred is a protein,

a) which has the ability to specifically bind with the EDb-a domain of fibronectin and contains α2β1-chain integrin,

b) which is specifically expressed or activated in endothelial cells,

in) which is specifically expressed or activated in stromal cells of the tumor,

g) which is specifically expressed or activated in tumor cells,

d) linking with EDb-a domain of fibronectin is amenable to inhibition by using the polypeptide and which contains α-chain integrin and

(e) the apparent molecular weight determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate ranges from 120 to 130 kDa for the light chain and 150 to 160 kDa heavy chain.

According to one preferred options endothelial cells are proliferating endothelial cells.

According to another preferred variant of the stroma is performance communications cells are stromal cells of the tumor.

Put in the invention the task is solved using a protein-specific binding with EDb-a domain of fibronectin mediates adhesion of endothelial cells, stromal cells of the tumor and tumor cells. In this case, the binding region can be characterized by the presence of at least one sequence selected from the group comprising SEQ ID nos: 1-3, and primarily contains α2β1-chain integrin.

Put in the invention the problem is solved then using a protein-specific binding with EDb-a domain of fibronectin induces proliferation of endothelial cells. In this case, the binding region can be characterized by the presence of at least one sequence selected from the group comprising SEQ ID nos: 1-3, and primarily contains α2β1-chain integrin.

Put in the invention the task is solved, in addition, using a protein-specific binding with EDb-a domain of fibronectin induces proliferation of endothelial cells in a collagen matrix, cell migration in collagen matrix and their differentiation in collagen matrix. In this case, the binding region can be characterized by the presence of at least one sequence selected from the group comprising SEQ ID nos: 1-3, and primarily contains α2β1-t the universi integrin.

Put in the invention the task is solved using a protein that binds to the EDb-a domain of fibronectin and induces specific pathways of signal transduction, when this is induced by at least one gene that encodes a protein selected from the group including

- focal adhesion kinase,

- D6-ligand (ALCAM),

- α-chain of the vitronectin receptor,

integrated alpha-8-subunit and

the predecessor related follistatin protein.

The region of connection in this case can be characterized by the presence of at least one sequence selected from the group comprising SEQ ID nos: 1-3, and primarily contains α2β1-chain integrin.

Preferably, when the induction of specific pathways of signal transduction at least one of these genes were induced at least once. It is preferable that at least one of these genes was induced twice.

Put in the invention the problem is solved then using antibodies having the ability to bind to offer in the invention of the protein.

Put in the invention the task is solved, in addition, by using antibodies having the ability to bind to a protein having the amino acid sequence selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3 and SEQ ID NO: 4.

In one of the preferred variants of the antibody has the ability to inhibit the effects that are specific to the EDbdomain.

Preferably, such binding and inhibition occurred in vitro and/or in vivo.

According to another preferred variant antibody is a monoclonal or recombinant.

In the following a preferred embodiment, the antibody is a scFv fragment.

Put in the invention the task is solved by using cells expressing the proposed invention is a protein.

In addition, put in the invention the task is solved by using cells expressing the proposed invention the antibody.

Put in the invention the problem is solved then using a phage expressing the proposed invention the antibody.

Put in the invention the task is solved, in addition, by using a method of screening compounds that bind to the receptor EDb-a domain of fibronectin, namely, that compare the response of cells in the presence of one or more of these compounds with the control response of the same cells in the absence of these compounds, however, such cells Express a proposed invention the protein or contain nucleic acid encoding the protein, and the response, respectively, to Strelna reaction mediated by receptor ED b-a domain of fibronectin.

According to one preferred options response, respectively, of the control reaction represents the adhesion of cells to surfaces that are covered with EDb-a domain of fibronectin or fragments.

In accordance with another preferred region linking EDb-a domain of fibronectin contains sequences SEQ ID nos: 1-4, or fragments thereof.

Preferably, the reaction, respectively, control the response of cells represented their proliferation on surfaces covered with EDb-a domain of fibronectin or fragments.

According to another one of the preferred variants of the reaction, respectively, control the response of cells represents the proliferation of endothelial cells in a collagen matrix, cell migration in collagen matrix and their differentiation in collagen matrix, such collagen matrix contains the EDb-a domain of fibronectin or fragments.

Connections proposed in the invention method is preferably selected from the group comprising antibodies, fragments of antibodies, artificial antibodies, peptides, low molecular weight compounds, aptamers and optical isomers.

According to another preferred variant antibodies are recombinant antibodies.

Ant the body is preferably selected from the group including scFv and its fragments.

Put in the invention the task is solved using the method of screening compounds that bind to the EDb-a domain of fibronectin, namely, that

a) cells are put in contact in the presence of different concentrations of one or more compounds present in a given protein concentration, which contains the EDb-a domain of fibronectin or a protein with the one presented in SEQ ID NO: 1-4 of the sequence, and

b) identify the differences between the observed in the presence of these compounds by the reaction of cells to the protein, which contains the EDb-a domain of fibronectin or a protein with the one presented in SEQ ID NO: 1-4 of the sequence, and the observed in the absence of these compounds control the response of cells to a protein that contains the EDb-a domain of fibronectin or a protein with the one presented in SEQ ID NO: 1-4 of the sequence, while these cells Express proposed in the invention is a protein or contain nucleic acid encoding this protein, and the reaction, respectively control the reaction cells is mediated by a receptor EDb-a domain of fibronectin.

While it is preferable that the reaction, respectively, control the response of cells represented their adhesion to surfaces coated with EDb-a domain of fibronectin or it is a fragment.

Monoclonal antibodies have been created by standard methods on hybrid technology and their characteristics were determined by immunohistological analysis obtained after deep cooling sections of human tumors (see Fig).

An example is the antibody (At) AM-EDBr-2 (mouse IgG 1/Kappa).

According to one preferred options response, respectively, control the response of cells represents their proliferation on surfaces covered with EDb-a domain of fibronectin or fragments.

According to another preferred variant of the reaction, respectively, control the response of cells represents the proliferation of endothelial cells in a collagen matrix, cell migration in collagen matrix and their differentiation in collagen matrix, such collagen matrix contains the EDb-a domain of fibronectin or fragments.

In the proposed invention the connection method is preferably selected from the group comprising antibodies, synthetic antibodies, antibody fragments, peptides, low molecular weight compounds, aptamers and optical isomers.

Put in the invention, the next task is solved through the use of a nucleic acid that encodes a protein that contains a sequence selected from the group comprising SEQ ID NO: 1-4, for selection of soy is ineni, which bind to the receptor EDb-a domain of fibronectin or EDb-a domain of fibronectin.

Put in the invention the task is solved through the application of the proposed invention the protein, respectively proposed in the invention antibodies for screening compounds that bind to the receptor EDb-a domain of fibronectin or EDb-a domain of fibronectin.

Put in the invention the task is solved in addition due to the application of the proposed invention the cells for screening compounds that bind to the receptor EDb-a domain of fibronectin or EDb-a domain of fibronectin.

Put in the invention the task is solved, in addition, through the use of a nucleic acid that encodes a protein that contains a sequence selected from the group comprising SEQ ID NO: 1-4, to generate antibodies or scFv fused proteins for use in diagnostic or therapeutic purposes.

Put in the invention, the next task is solved through the application of the proposed invention the protein for generating antibodies or scFv fused proteins for use in diagnostic or therapeutic purposes. The term "therapeutic use" means, among other things, directed against angiogenesis treatment and the use of compounds, which inhibit specific interaction between the EDband its receptor. In this case discusses the antibodies to the receptor, and to the EDbwhen this is used peptides with the sequence SEQ ID NO: 1-3 and their stable derivatives and low molecular weight compounds.

Put in the invention the task is solved through the application of the proposed invention the cells to create antibodies or scFv fused proteins for use in diagnostic or therapeutic purposes.

Put in the invention the task is solved in addition due to the use proposed in the invention of the phage to generate antibodies or scFv fused proteins for use in diagnostic or therapeutic purposes.

Put in the invention, the next task is solved through the use of a protein containing a sequence selected from the group comprising SEQ ID NO: 1-4, for Pro-angiogenic therapy.

Put in the invention the task is solved through the use of a protein containing a sequence selected from the group comprising SEQ ID NO: 1-4, for diagnostic purposes.

Put in the invention the task is solved through the use of a protein containing a sequence selected from the group comprising SEQ ID NO: 1-4, for gene therapy.

Postavljena the invention the problem is solved, in addition, through the use of a protein containing a sequence selected from the group comprising SEQ ID NO: 1-4, to cover the surfaces that contact the endothelial cells.

While it is preferable that such coverage has been in vitro or in vivo.

Put in the invention the task is solved through the use of a protein containing a sequence selected from the group comprising SEQ ID NO: 1-4, in cell cultures.

Put in the invention, the next task is solved through the use of a protein containing a sequence selected from the group comprising SEQ ID NO: 1-4, together with at least one graft.

Such a graft is preferably selected from the group including vessel(s), skin, cornea, kidney, liver, bone marrow, heart, lungs, bone, thymus, small intestine, pancreas, and other internal organs, as well as their parts and cells.

Put in the invention the task is solved through the use of a protein containing a sequence selected from the group comprising SEQ ID NO: 1-4, together with at least one implant.

This implant is preferably chosen from the group comprising lung implants, pacemakers, artificial heart valves, implants vessel, implants, screws, tires, plates, wire, the impact, rods, artificial joints, breast implants, artificial cranial plates, artificial teeth, dental fillings and dental bridges.

The term "impacts specific to the EDb-a domain of fibronectin", refers to all those effects are due to the EDb-a domain of fibronectin, but not EIII7, EIII8 etc. One of these impacts is described, for example, Chen et al, 1998 (see above), i.e. it is the rapid tyrosine phosphorylation of a large number of intracellular proteins in contrast to the slower phosphorylation in adhesion, mediated by domains EIII8-9-10. By "low molecular weight compounds" refers to all those connections, the relative molecular mass of less than approximately 1000-1200. By "aptamer" refers consisting of nucleic acid molecules that are capable of performing the function of highly specific ligands for a large number of biomolecules. The term "Pro-angiogenic therapy" refers to any form of therapy in which you want to stimulate angiogenesis. The term "directed/directed against angiogenesis treatment/therapy" refers to any form of treatment/therapy, the purpose of which/which is the inhibition of angiogenesis. Under "gene therapy" refers to any form of therapy, which aims to turn off the tion is caused by a gene defective functions, accordingly, the restoration of normal function of the gene diseases that can be affected by the elimination or, on the contrary, the introduction of a protein. Such therapy may include, but not limited to, the introduction of foreign DNA into somatic cells. The term "cell culture" refers to the media for cell cultures, as well as vessels for cell culture. Such vessels for cell culture preferably include flasks, cups, wells and plates for cell culture, tetralonia microplates, 96-well plates, flasks for cell culture and bioreactors.

Under the term "diagnostic" refers to all targets that are used to detect the condition of the body/organ/cell, respectively, for defining the actual state of the body/the body/cells to a certain category of States (for example, to a specific disease)that, for example, involves the use of (but not limited to) a specific set/chemicals/measuring device for determination of some physical quantities, such as temperature, etc. or chemical parameter, such as concentration, etc.

The term "therapeutic use" refers to all targets, which serve to facilitate, respectively, is zlecenia painful condition of the body/the body/cells. Under the concept of "use of protein together with the implant" refers to combined either in time or in space application. So, for example, protein molecules can be mobilitat on the implant with him to implant into the body or you can enter them in the body of the spatial separately from the implant, but at the same time with his "implantation" (injection and so on).

Below the invention is described in more detail based on examples with reference to the accompanying drawings on which is shown:

figure 1 - schematic representation of repeated sequences of type III, which are used in the research according to the invention,

figure 2 - results of analysis of proliferation under the influence of the EDb-a domain of fibronectin (ED-B) on endothelial cells, respectively, on the human stromal cells using different substrates,

figure 3 - test results overgrowth (test on the formation of the "tubes") of endothelial cells under the influence of ED-B,

figure 4 - test results adhesion (adhesion), which explored the adhesion of endothelial cells to surfaces coated with ED-B,

figure 5 - test results, similar to the test, the results of which are presented in figure 4, except that the cells were subjected to pre-incubation with various synthetic peptides, is posledovatelnosti which was a partial sequence of the ED b-a domain of fibronectin,

figure 6 is a partial sequence synthetic peptide EDb-a domain of fibronectin used in the test, the results of which are presented in figure 5,

figure 7 - results of test for adhesion of endothelial cells to different synthetic ED-B-peptides

on Fig - location of the synthetic peptides indicated in Fig.6-7, on the model of the structure of the main peptide chain ED-B,

figure 9 - the impact of the EDb-a domain of fibronectin and located on the loop 5 peptide (SEQ ID NO: 2) on the formation of capillarity structures identified in the test for growth of cells (test on the formation of the "tubes"),

figure 10 - results of two analyses are carried out using affinity chromatography using Fn-clamps 7-8-9, respectively Fn-7-B-8-9 obtained from cell lysates of endothelial cells of human skin, bearing the label on the outside

figure 11 - results of two analyses are carried out using affinity chromatography using Fn-clamps 7-8-9, respectively Fn-7-B-8-9 obtained from cell lysates stromal cells of human skin, bearing the label on the outside

on Fig - cleaning results EDbB-receptor with affinity chromatography and

on Fig images obtained after deep cooling slices of human op is Holi, characteristics are determined by immunohistological analysis.

Figure 1 shows the various used in conducted in accordance with the present invention study of recombinant fragments of fibronectin, which are the domains of different structures with different repeated sequences of type III. Thus Fn-7-B-8-9 includes domains 7, EDb8 and 9, Fn-clamps 7-8-9 includes domains 7, 8 and 9, ED-B includes domains EDb, Fn-10 includes domain 10, a Fn-6 includes domain 6 fibronectin. These proteins are expressed in E. coli in the form of labeled using His-Tag protein and was purified on a Nickel-chelate-sepharose column. Used in this study, numerical designations correspond to those used in the scientific literature. Thus, each of abbreviations FN-B, ED-B, EDB and EDbrefers to the EDbdomain of fibronectin and they should be treated as synonymous.

Figure 2 presents the results of the analysis of proliferation, which explored the impact of the EDb-a domain of fibronectin (ED-B) on the proliferation of endothelial cells (EC), respectively stromal cells (SC). In each well of 96-hole tablet was introduced at 1000 cells and incubated. In the analysis of cell proliferation in medium were added soluble ED-B (10 µg/l). After three days the number of cells was determined using the MTS assay. The proliferation of CL is the current induced through the primary growth factor fibronectin (bFGF). It was found that ED-B had no effect on cells in the absence of bFGF, and did not reveal any effect on the cells belonging to type III domain 10 of fibronectin in the presence of bFGF (data not shown). The impact of the ED-B on the proliferation of human endothelial cells was detected for cells seeded on gelatin (EC/gelatin), and for cells seeded on collagen (EC/collagen), but in the last case the effect was not as pronounced as for cells seeded on gelatin. In the case of human stromal cells seeded on gelatin (SC/gelatin), their proliferation has occurred even in the absence of bFGF and its level was significantly higher than the level of proliferation of human endothelial cells. The addition of bFGF, bFGF, respectively+ED-B did not lead to increased proliferation. As a measure, on the basis of which was determined by the number of cells was measured by optical density at a wavelength of 490 nm.

For analysis of proliferation used the following experimental method.

Material:

96-well plate (flat bottom), the firm Nunc.

Environment:

MCDB 131 supplemented with penicillin/streptomycin (Pen/Strep), amphotericin (0.25 microgram/ml), heparin (20 μg/ml), inaktivirovannye heating FCS (fetal calf serum) (5%).

Technique:

Cells from the calculation of 500-1000 cells per well (96-well what lansey) for 3 days cultured in 100 μl of medium with bFGF (1-3 ng/ml) or VEGF (30-50 ng/ml). The exact number for each portion should be determined by titration: the optimum is the minimum concentration, which provides maximum stimulation of cell proliferation. Synchronization of cells before the experiment is not required, but acceptable. After 3 days the number of cells is determined using the set for MTS-analysis (firm Promega)following the manufacturer's instructions. To identify the maximum absorption in the linear region the absorption is recommended to measure at several points in time (0, 5, 1, 2, 4 h).

Controls:

negative control without mitogen (proliferation missing) (-bFGF/VEGF);

positive control in the presence of mitogen (maximal stimulation) (+bFGF/VEGF).

Figure 3 presents data describing the impact of the ED-B on the growth of endothelial cells from spheroids. In this experiment, HUVEC-spheroids (from the English. Human Umbilical Vein Endothelial Cells, the endothelial cells of human umbilical vein) were placed ("introduce") in collagen and adding 10 μg/ml bFGF (basic fibroblast growth factor) induced their proliferation in the presence of 6 μg/ml ED-B with or without him. It was found that the addition of only one bFGF induces overgrowth, which is then optionally be enhanced by the addition of ED-B (+bFGF+ED-B).

In the test for growth (test on the formation of the "tubes") used the trail is in store experimental method.

Material:

the methylcellulose with a maximum viscosity (firm Sigma);

trypsin/etc for cell culture (company Gibco);

round-bottom 96-well plates (firm Greiner, cat. No. 650185)

recombinant bFGF (company Gibco, cat. No. 13256-029);

recombinant VEGF (firm R & D System);

antikrizisnyi CD31 (firm RDI, cat. No. RDI-CD31TLD);

heparin (company Gibco, cat. No. 15077-027).

Solutions:

SFR/antibiotics: cell culture-SFR, 10x penicillin/streptavidin (Pen/Strep), 2.5 µg/ml amphotericin b;

1%gelatin (Difco company), autoclave and, after cooling, mixed with penicillin/streptavidin (Pen/Strep) and amphotericin (0.25 microgram/ml).

Environment:

MCDB 131, glutamine, penicillin/streptavidin (Pen/Strep), amphotericin (0.25 microgram/ml), heparin (20 μg/ml), inactivated by heating FCS (10%).

Environment for growth:

medium with 2 ng/ml bFGF and 10 ng/ml VEGF.

Cells:

HUVEC;

MVEC skin (the number of passages >4).

Technique:

Endothelial cells are separated using trypsin/etc and diluted in containing 0,24% methylcellulose medium to a concentration of 5000 cells/ml In each well of pen Greiner make 200 ál (1000 cells) and incubated over night. Round clusters of cells (spheroids) are collected using a 1-ml pipette with a cut tip and separated by centrifugation. Spheroids resuspending 1.2%mixture of methylcellulose in FCS) and the t with neutralized collagen gel. Thus there is a co-EDband bFGF.

Shown in the drawing, the data suggest that adding ED-B overgrowth of endothelial cells markedly intensified and exceeds the level induced by bFGF.

Figure 4 shows the test results on the adhesion of endothelial cells to tetrazinni the microplate coated with ED-B. In this experiment, the endothelial cells that were in the original vessel for their cultivation, were separated from their substrate by treatment with trypsin (trypsin/etc) and then incubated in titration microplate coated with ED-B in various concentrations(0, 1, 2, 3, 5, 10, 20, 40 µg/ml), and left for one hour for adhesion. As a negative control was used wells coated with BSA (bovine serum albumin) at a concentration of 1 mg/ml; from the data obtained in the experiment, read the level of adhesion to BSA (<10%).

The degree of adhesion was quantitatively assessed by crystal violet staining, after which he caused lysis using ordinator. Quantitative evaluation was performed by measuring the optical density at a wavelength of 595 nm. The horizontal line drawn on the graph when the value of A595nm≈1,06 corresponds to 100%adhesion in the plasma fibronectin.

The results of this experience suggests that sticking to etoc covered in ED-B surfaces due to the presence of the cell surface receptor ED-B domain.

To test the adhesion/adhesion used the following experimental method.

Solutions:

1%BSA (firm Sigma, precipitated with ethanol);

2%solution of serum in SFR (or trypsin neutralizing solution).

Environment:

MCDB 131, penicillin/streptavidin (Pen/Strep), amphotericin b (2.5 µg/ml), heparin (20 μg/ml), 0.1%BSA, Sigma, precipitated with ethanol);

0.1%crystal violet, 2%glutaric aldehyde in SFR, sterilized by filtration;

2% - ordinator.

Technique:

To the wells of the 96-hole tablet (firm Nunc) at 37°To contribute one hour protein. When using low molecular weight proteins (<20 kDa) or peptides, it is recommended to let them dry out on tablets (over night without a cover in a sterile environment). Then the wells for 1 h at 37°saturate With 1%BSA. Cells are separated by a single trypsin, washed with 2%solution of serum to inactivate trypsin and resuspended in the environment. If you need to test antibodies or peptides, the cells were pre-incubated with them in suspension for 30 min at 37°C. To each well, incubated for 104cells (using 96-well plate) in a volume of 50-100 µl for 1 h at 37°C. the Supernatant is carefully poured, tablets for Oceania of them remnants of the liquid in the inverted position set is more for 1 minute on a paper towel and then adherent cells for 15 min dye crystal violet/glutaraldehyde and fixed. The wells are washed three times SFR and then the cells are lysed by adding 2%LTOs (within 15 min using Shuttle device). The absorption is measured at a wavelength of 595 nm. If necessary, cells after triple rinsing water can be painted again.

Controls:

negative control - blank wells (BSA control);

the positive control contained in plasma fibronectin (2.5 µg/ml).

Adhesion calculated as follows:

% adhesion=A595(sample): 100×And595(fibronectin).

Figure 5 presents the results of the test, which is similar to the results presented in figure 4, except that before the binding is covered with ED-B tetrazinni the microplate endothelial cells were pre-incubated with various synthetic peptides at a concentration of 250 μm, the sequence of which was a partial sequence of the EDb-a domain of fibronectin. Binding was determined by measuring optical density at a wavelength of 595 nm (A595). Refer to peptides that are specified on the drawing and are explained in Fig.6. When this peptide sequence No. 043 corresponds to the sequence presented in SEQ ID NO: 1, peptide sequence No. 553, corresponds to the sequence SEQ ID NO: 2, a peptide sequence No. 038 STA is esthet sequence SEQ ID NO: 3. A higher value And595corresponds neighbormedia adhesion of cells, whereas a lower value And595corresponds to the inhibitory effect of the corresponding peptide in adhesion.

The study was performed according to the method described for figure 4.

Figure 6 presents selected from the full sequence EDb-a domain of fibronectin partial sequence synthetic ED-B-peptides along with chosen for them by the symbols of the sequences. To denote the amino acid one-letter code is used.

Figure 7 presents the results of the test, which is similar to the results shown in figure 5, except that in this case the wells titration microplate was covered not EDb-a domain of fibronectin, and pre-incubated with peptides, which, according to the results of the experiment are presented in figure 5, have proven themselves as having, respectively, do not possess inhibitory activity, and in this way was coated wells of these peptides. While on the measured value And595it was found that the adhesion of cells in this experiment occurred in the floor of one of the holes having the inhibitory activity of the peptides, whereas the use of the peptide according to the results of the experiment are presented in figure 5, Zarco who was andoval themselves as not possessing inhibitory activity adhesion of cells occurred.

The study was performed according to the method described for figure 4.

On Fig presents the model structure of the EDb-a domain of fibronectin (ED-B), on the basis of which it is possible to determine the location of possessing inhibitory activity of peptides No. 1 (= SEQ ID NO: 1), №2 (= SEQ ID NO: 2) and # 3 (=SEQ ID NO: 3). Obviously, these possessing inhibitory activity of the peptides are located on loop 1, respectively, the loop 5 of the structure of ED-B, and thus correspond to those regions of the domain, through which there is a binding to the cell, respectively located on the cell receptor. Presented at Fig model of the structure of the ED-B domain based on already identified the structure fibronectin domain 7 type III. N-T and s-T denote the N - and C-end, respectively.

Figure 9 presents the results of the experiment, which explored the effects of the addition of ED-B domain and previously identified as having inhibitory activity of peptide No. 2, and add fibronectin domain 6 type III on the induction capillarity structures (formation of "tubes") in the test for growth. It was found that, compared with baseline, bFGF induced by the penetration of the cells in the collagen gel greatest impact on penetration is exerted inhibitory Prilepin the e peptide, having the sequence of SEQ ID NO: 2. This peptide has also been a stimulating effect on the penetration of endothelial cells in collagen gel. Thus, this peptide corresponds to the connecting areas EDbdomain and similarly most EDbdomain stimulates the penetration of endothelial cells in collagen.

The study was performed according to the method described for figure 3.

Figure 10 presents the results of affinity chromatography lysate labeled on the surface of endothelial cells of human skin. To this end proliferating, endothelial cells treated with Biotin surface was literally using detergent and subjected to affinity chromatography, in which short fragments of fibronectin containing or not containing typed in them EDb-a domain of fibronectin, was associated with separate (version with EDb-a domain of fibronectin denoted as Fn-7-B-8-9, a variant without EDb-a domain of fibronectin denoted as Fn-clamps 7-8-9). According to the results of chromatography, it was found that biotinylated protein with an apparent molecular weight of 120-130 kDa specifically binds to contain ED In the fragment (marked by arrow). Elution was performed using etc. It was collected several fractions, which are described below. Then these fractions were subjected LTO-PAG-electrophoresis and analysed is ovale using Western blotting using streptavidin-peroxidase and chemiluminescence (ECL). Lanes 1 and 5 correspond to pre-elyuirovaniya fractions and lanes 2, 3, 4, 6, 7, 8 correspond elyuirovaniya fractions 1, 2 and 3. Lanes 1-4 obtained by chromatographytandem using Fn-clamps 7-8-9, and lanes 5-8 received chromatographytandem using Fn-7-B-8-9. Shown in the drawing the chromatography results convincingly show that the most pronounced band attributable to the molecular weight of 120-130 kDa, corresponds to a protein that specifically binds to contain the EDbfragment of fibronectin and thus represents a receptor-EDb-a domain of fibronectin.

For biotinidase and lysis of endothelial cells used the following experimental method.

Material:

3 sulfo-N-hydroxysuccinimidyl ether of biotinidase acid, the firm Sigma,

SFR with/without Mg/Ca (firm Dulbecco),

the HEPES buffer: 20mm HEPES (2-N-hydroxyethylpiperazine-N'-2-econsultancy acid) with a pH of 7.6, 1 mm CaCl2, 1 mm MgCl2, 0.1%NaN3,

1%CHAPS (about./about.) and

included in this kit Mini-prep company Boehringer complete protease inhibitor not containing add, cocktail tablets.

Technique:

Cup for cell culture before bioterrorism and after each time, three times washed SR without Ca+Mg. Before the last wash add biodynamy buffer (1 mg per 15 ml SPR). In which R each Cup slowly with a pipette and add a buffer in an amount of 5 ml (for cups with the bottom area of 225 cm 2) or 12.5 ml (for cups with the bottom area of 500 cm2)that allows for the swinging cups from side to side to evenly distribute across their bottom made in them. Then the contents of the first Cup handle half of the total volume of the buffer for lysis. This buffer with a pipette and contribute to the Central part of the bottom of the Cup and spread across its surface. After that, cells are scraped with the help of the knife. Then all the contents of the 1st Cup for cultivation with a pipette and transferred to the 2nd Cup, in which I repeat the entire above process. From the last Cup all its contents are transferred into 50-ml conical centrifuge tube. All the above process is repeated in all the cups for cultivation using the second half of the buffer for lysis (without scraping cells) and the final volume is also transferred into a centrifuge tube. Next, the contents of the 50-ml conical centrifuge tubes, centrifuged at room temperature (RT) for 5 min at 3000 rpm (tabletop laboratory centrifuge of Heraeus). The lysate is collected with a pipette and ideally immediately use in affinity chromatography if necessary, however, it can also be stored in a frozen state at -80°).

For covalent binding of the s protein with separate used the following method.

Material:

activated sepharose CH Sepharose 4B company Pharmacia Biotech, code No. 17-0490-01,

1 mmol of HCl, 2,2%NaHCO3.

Technique:

HCl cooled in an ice bath, and separate allowed to warm to room temperature. Then sepharose washed with 1 mmol of HCl. 1 ml sepharose you want to use 10 ml of HCl. Sepharose give gently to drain into a pre-chilled tube, where it then swells for about 15 min (1 g sepharose corresponds to 3 ml of the swollen sepharose). Then the test tube is centrifuged for 1 min at 800 rpm the Supernatant is collected by pipette and discarded.

This process was repeated three times.

After the 3rd wash again add HCl, the tube is shaken and centrifuged for 3-5 min at 800 rpm the Supernatant is collected with a pipette and obtained after centrifugation the precipitate in the test tube was dissolved in 20 ml filtered through millionby water filter and transferred into two new centrifuge tubes (in each case, one tube is used for 7-EDB-8-9-sepharose and clamps 7-8-9-sepharose, i.e. separate, which binds to the polypeptide having the replays III7, EDb, III8 and III9, respectively III7, III8 and III9). Immediately after this, the tubes centrifuged again and the supernatant is collected with a pipette, linking in this way 1-5 mg protein/ml sepharose (i.e. 2 mg protein/ml 78-9-sepharose;

2 mg protein/ml 7-EDB-8-9-sepharose)

The contents of the tubes are mixed by inverting. Then continuously add 2,2%NaHC3(50 µl/ml of gel). The result is the neutralization of the residual HCl. The tubes are turned over and their contents with maximum intensity is stirred for 1-5 h at "swing table".

The contents of the tubes centrifuged again.

To determine the concentration of the protein in which it should be used for covalent binding with separate, conducted a test of Bradford.

Material:

the mother solution of BSA at a concentration of 2 mg/ml,

the Bradford reagent.

Technique:

The BSA solution is applied to the tablet for immunoassay (type Maxi Sorp firm Nunc) as follows: 5 ug-4 ug-3 ug-2 ug-1 ug (volume of 80 ál+20 ál of the test material).

Pre-breeding BSA: 5 ág/50 ál=0.1 mg/ml

The mother solution with a concentration of 2 mg/ml is diluted by a dilution ratio of 1:20 to a concentration of 0.1 mg/ml

Affinity chromatography, respectively elution was carried out as follows.

a) Affinity chromatography

Material:

activated sepharose CH Sepharose 4B company Pharmacia Biotech, code No. 17-0490-01,

buffer A (20mm HEPES with pH of 7.6, 1 mm CaCl2, 1mm MgCl20.1%NaN3),

buffer B (buffer A+150 mm NaCl+0.1%CHAPS),

buffers (buffer A+0.1%CHAPS),

buffer with pH 4 (filtered through millionby filter water+0,1%glacial acetic acid+0.1%CHAPS),

Add-buffer (buffer A+200mm etc with a pH of 8.5+0.1%CHAPS).

Technique:

The lysate first three times through the column.

Under the column have a test tube for collecting fluid. The first 2 ml of the lysate gently with a pipette and transferred to Eppendorf gel. For other portions of the lysate using a measuring pipette. One should especially pay attention to the fact that the speaker must take a strictly vertical position. When you first use the column prior to production run, conduct a "dummy run" using all containing no protein buffers. The packing of the column should be used a maximum of 5 times.

If using frozen lysate (-80° (C), it is first heated using a water bath and then centrifuged (5 minutes at 3000 rpm).

But in any case, it is preferable to use fresh, not frozen lysate.

From the lysate using the pipette, the sample volume of 500 ál and make it into the flask Eppendorf. This sample is used to study the lysate before and after chromatography.

When using 2 columns (one for clamps 7-8-9-sepharose, and the other for 7-In-8-9-sepharose) through each of them miss half of the total lysate. The flow rate through both the colon is and must be the same. Failure to comply with this condition more "slow" column is kept closed during the relevant period of time. The optimum flow rate is 0.2-0.5 ml/min

After triple pass lysate through the column from the resulting product after mixing with a pipette and in this case also selected a sample volume of 500 ál, which is placed in the flask Eppendorf for adequate investigation of the lysate before and after chromatography.

In the end passed through the column buffer B and buffer volume, which in each case corresponds to 10 times the volume of the column. This washing process is completed.

b) Elution

Preliminary elution: passed Through the column buffer In order to establish whether in the column despite her washing any proteins. 500 ál released from the column fractions collected in the flask Eppendorf. (When using 2 columns respectively collect 2×500 ál).

Elution with add: add forms a complex with ions of CA and Mg. This results in the elution of proteins of endothelial cells to bind them (proteins) needed CA and Mg. 4 ml add buffer twice passed through the column (respectively through both the column and is collected in two fractions (E1 and E2/WE and VE) in a test tube "Falcon". Then the contents of the test tubes premesis the Ute with a pipette and transferred to a 5000 ml one (respectively two) flask(s) Eppendorf.

Elution at pH 4: Own pH value of the buffer is 3.7. Outside the neutral pH range (pH 6-8) binding of the receptor with its corresponding protein can inhibit. In this case, as with elution using add, through the column twice passed in 4 ml of buffer with pH 4, released from the column material is collected in two fractions and using the pipette 500 ál of each of them (for 4.1 and 4.2/4.1 and 4.2).

In the end passed through the column buffer And the volume corresponding to the 3rd volume of the column, washing out the way of the speakers acid. The last portion of the buffer in a volume corresponding to the volume of the column, left column. The column is closed and placed in storage in the refrigerator.

Fractions with a volume of 500 µl frozen in Eppendorf tubes, soaking for at least 15 min at -80°C, and then subjected to freeze-drying device type "Speed vac".

Thus obtained fractions, respectively, pre-erwerbende fraction, share with LTO-PAG electrophoresis and analyzed by Western blotting in reducing conditions.

Figure 11 presents the results of an experiment similar to the experiment whose results are shown in figure 10, except that in this case used not lysed endothelial cells,and stromal cells lysed. Figure 11, which shows the results of the analysis by the method of Western blot, lanes 1-3 correspond to products, elyuirovaniya of columns for affinity chromatography using Fn-clamps 7-8-9, and lanes 4-6 correspond to products, elyuirovaniya of columns for affinity chromatography using Fn-7-B-8-9. Lanes 1 and 4 correspond to the pre-elyuirovaniya fractions and lanes 2, 3, 5, 6 correspond to fractions 1 and 2 corresponding buervenich straps. This cell lysate derived from human stromal cells, missing most evident band, which falls on the molecular weight of 120-130 kDa and which can be found in figure 10.

Considered in the foregoing description, in the claims and represented in the drawings, the distinctive features of the invention in various embodiments can be essential to the invention, the value individually and in any combinations of these.

On Fig presents data for linking the ED-B domain of protein, which was purified by affinity chromatography by the method described above and were separated by gradient gel electrophoresis in the presence of LTOs (from 4 to 12%). Areas with specific accumulation, which correspond to the dual-band (indicated by arrows), cut out and analyzed using mass spectroscopy.

In the cut is litate the sequencing of the isolated protein was able to be unambiguously identified as A2-beta-integrin, in which the dominant heavy chain corresponds beta-and light chain corresponds to the A2-subunit.

The results obtained indicate that the binding EDbdomain mainly mediated beta-subunit of integrin. Depending on the investigated cell type other alpha-subunit (e.g., A2) in combination with beta-subunits can also mediate binding to the EDB-FN.

On Fig presents the results of immunohistological analysis with deep freeze slices of human tumors, where the following symbols are used:

A: renal cell cancer, arrows indicate specific staining using At AM-EDBr-2;

B: enlarged image of the same drug;

In: hepatic cell carcinoma;

G: melanoma (in this case, is not revealed specific staining).

Analysis EDB-receptor

Strips cut from the 1D-gel, washed with a solution of NH4HCO3and acetonitrile, dried and proteolysis of proteins in the gel were mixed with a solution of trypsin. Erwerbende of the gel solution to decompose ("digestion"), the peptides were concentrated on μC18-columns absoluely and analyzed using MALDI-mass spectrometry (obtaining a list of the masses of the peptides "digested" protein).

On the basis of the detected masses PE is the Chida from each cut from the gel strip was conducted the search in the data Bank. In that case, if the search was received with mixed results, for a single peptide addition was measured by MALDI-PSD spectra (fragment spectra). These spectra are either directly used to confirm the expected peptide sequence (interpretation of the spectrum), or on the basis of this spectrum conducted a search in the database.

The investigated bands:

Band=band 1 from preparation 6

band 4 drug 5

band 6, obtained by elution acid

Results: integrin α2

see the search results in the data Bank for strip 4

spectra obtained for bands 1 and 6, indicate the presence of peptides, giving the same intensity

- PSD spectrum of a peptide from a strip of 1 indicates that a partial sequence corresponds to the integrin α2

Band B= band 2 from preparation 6

band 5 from preparation 5

band 7, obtained by elution acid

Results: integrin β1

see the search results in the data Bank for bands 5 and 7

- spectrum obtained for strip 2, indicates the presence of peptides, giving the same intensity

the results of the search in the database using the PSD spectrum band 2 confirmed its compliance with the integrin β 1

BSA

- is present in all three bands

- presence confirmed the results of the search in the database using the PSD spectrum and mass of numerous peptides.

1. Method of screening compounds that bind to the receptor EDb-a domain of fibronectin, namely, that compare the response of cells in the presence of one or more of these compounds with the control response of the same cells in the absence of these compounds, however, such cells Express a protein that has the ability to specifically bind with the EDb-a domain of fibronectin and which contains α2β1-chain integrin, or contain nucleic acid encoding the protein, and the response, respectively, of the control reaction, mediated by a receptor EDb-a domain of fibronectin.

2. The method according to claim 1, where the reaction of, respectively, the control reaction, is the adhesion of cells to surfaces that are covered with EDb-a domain of fibronectin or fragments.

3. The method according to claim 1 or 2, characterized in that region linking EDb-a domain of fibronectin contains any sequence selected from the sequences presented in SEQ ID nos:1-4, or fragments thereof.

4. The method according to claim 1, characterized in that the reaction of, respectively, the control reaction, cells represents their proliferation on surfaces that are covered with EDb-a domain of fibronectin or fragments.

5. The method according to claim 1, characterized in that the reaction, respectively, control the reactions is, cells represents the proliferation of endothelial cells in a collagen matrix, cell migration in collagen matrix and their differentiation in collagen matrix, such collagen matrix contains the EDb-a domain of fibronectin or fragments.

6. The method according to any one of claims 1 to 5, wherein the compound is chosen from the group comprising antibodies, synthetic antibodies, antibody fragments, peptides, low molecular weight compounds, aptamers and optical isomers.

7. The method according to claim 6, characterized in that the antibodies are recombinant antibodies.

8. The method according to claim 6, wherein the antibody is chosen from the group comprising scFv and its fragments.

9. Method of screening compounds that bind to the EDb-a domain of fibronectin, namely, that

a) cells are put in contact in the presence of different concentrations of one or more compounds present in a given protein concentration, which contains the EDb-a domain of fibronectin, and (b) identify the differences between the observed in the presence of these compounds by the reaction of cells to the protein, which contains the EDb-a domain of fibronectin, and observed in the absence of these compounds control the response of cells to a protein that contains the EDb-a domain of fibronectin,

in addition, these cells Express the protein of the one presented in SEQ ID NO:1-4 of the sequence or chain α 2β1 integrin, or contain nucleic acid encoding this protein, and the reaction, respectively control the reaction cells is mediated by a receptor EDb-a domain of fibronectin.

10. The method according to claim 9, in which the reaction is correspondingly control the reaction cells is their adhesion to surfaces coated with EDb-a domain of fibronectin or fragments.

11. The method according to claim 9, characterized in that the reaction of, respectively, the control reaction, cells represents their proliferation on surfaces that are covered with EDb-a domain of fibronectin or fragments.

12. The method according to claim 9, characterized in that the reaction, respectively, control the response of cells represents the proliferation of endothelial cells in a collagen matrix, cell migration in collagen matrix and their differentiation in collagen matrix, such collagen matrix contains the EDb-a domain of fibronectin or fragments.

13. The method according to any of PP-12, in which the compound is chosen from the group comprising antibodies, synthetic antibodies, antibody fragments, peptides, low molecular weight compounds, aptamers and optical isomers.

Priority items:

07.09.2000 according to claims 1 to 13;

02.05.2001 according to claims 1-13.



 

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4 tbl

FIELD: medicine.

SUBSTANCE: method involves determining plasminogen, fibrinogen and fibrin degradation products level and blood platelets aggregation and disaggregation values in treating a patient. Blood platelets aggregation rate growing twice as high at the fifth day, blood platelets disaggregation growing 5-8 times as high at the third-fifth day, plasminogen concentration growing 1.5 times as high at the eighth day and fibrinogen and fibrin degradation products level dropping 2.5 times as low at the eighth day, favorable treatment outcome is to be predicted. Aggregation rate dropping twice as low at the fifth day with blood platelets disaggregation being lost at the eighth day and no fibrinogen and fibrin degradation products level changes being detected, unfavorable prognosis is considered to be the case.

EFFECT: higher accuracy of early stage prognosis.

2 tbl

FIELD: medicine.

SUBSTANCE: method involves determining lactate, pyruvic acid concentration, superoxide dismutase activity in patient saliva sample. Lactate concentration increase being equal to or and higher than 50%, pyruvic acid concentration drop being equal to or greater than 60% and superoxide dismutase activity being equal to or and higher than 50% when compared to norm, neurocirculatory dystonia diagnosis is to be set.

EFFECT: simplified noninvasive diagnosis method in preclinical stage cases.

FIELD: clinical medicine, laboratory diagnostics.

SUBSTANCE: one should carry out complex laboratory survey including daily urinary sampling by Zimnitsky and blood sampling for analysis just after finishing sampling the last urinary portion. In every urinary portion one should detect pH, protein and relative density, in total urinary volume it is necessary to detect concentration and quantity of uric acid, creatinine and daily proteinuria, calculate clearances of uric acid and creatinine and fractional clearance of uric acid. On detecting purine exchange disorders including high concentration of urinary uric acid at low daily diuresis one should recommend to widen drinking mode; at altering urinary reaction towards acidic side one should prescribe alkalizing preparations; in case of pronounced purine exchange disorders and severe accompanied pathology one should carry out extracorporal therapy. The innovation enables to considerably improve the quality of diagnostics and match the most optimal schemes of therapy.

EFFECT: higher efficiency of therapy.

5 cl, 3 dwg, 5 ex, 20 tbl

FIELD: biochemistry, in particular method for determination of antioxidant enzyme superoxide dismutase (SOD) activity.

SUBSTANCE: claimed method includes preliminary determination of appropriate xanthine oxidase activity (E3) in examined sample, namely examined sample is added into dish to incubation medium containing 50 mM sodium carbonate solution; 0.1 mM EDTA solution; 37.5 muM nitroblue tetrasolium (NBT) solution in 50 mM phosphate buffer (pH 10.2); and 73 mul of 0.1 mM xanthine solution in 0.5 N NaOH and free from xanthine oxidase. Optical density variation is registered for 3-5 min at 560 nm wavelength using obtained kinetic curve. (E3). Initial velocity of NBT reduction without SOD is determined according to known method. In the same dish examined sample is added, wherein said sample contains SOD in amount sufficient to change solution optical density caused by NBT reduction at rate of 0.015-0.02 U/min and selected for one-type samples under conditions wherein said variations are occurred at substrate excess reduction in proportional variations of reaction velocity. Optical density variation per one minute in terms of protein amount in sample is calculated and protein amount in examined sample is determined. SOD activity is calculated according to specific formula.

EFFECT: method of increased accuracy and reliability.

FIELD: biochemistry.

SUBSTANCE: invention relates to method for biological material sample analysis in biological or immunological tests, wherein in sample treatment process coloring is observed, and color darkening is correlated with amount of tested substance. At least one color characteristic, such as coloring angle, chromaticity, darkening, and obtained color brightness is measured. Method of present invention is useful in analysis of lung, throat, cervix uteri or spermatic liquid mucilage for diagnosis of cancerous and precancerous conditions.

EFFECT: enhanced assortment of agents for diagnosis of cancerous and precancerous conditions.

14 cl, 4 dwg

FIELD: medicine.

SUBSTANCE: method involves analyzing whole capillary blood on monocytosis and lymphocytosis in the cases the depression is clinically apparent according to Hamilton scale at 22.1±2.3 points determining infrared blood spectroscopy absorption indices in infrared spectral analyzer during 30 s in bandwidth ranges of 3085-2832 cm-1, 1543-1425 cm-1, and 1087-963 cm-1, and determining mean absorption indices. The mean absorption indices being found equal to 40.2±2.0%, 38.0±3.2%, 43.1±1.9%, in bandwidth ranges of 3085-2832 cm-1, 1543-1425 cm-1, and 1087-963 cm-1, in combination with normal monocytosis and lymphocytosis, respectively, depression in remote mild craniocerebral injury period is to be diagnosed. The mean absorption indices being found equal to 49.5±2.6%, 54.4±3.2%, 47.8±2.8%, in bandwidth ranges of 3085-2832 cm-1, 1543-1425 cm-1, and 1087-963 cm-1, in combination with monocytosis > 5% and lymphocytosis >25% in leukocytic formula of bulk blood analysis, respectively, therapeutically resistant depression in remote mild craniocerebral injury period is to be diagnosed.

EFFECT: high accuracy of diagnosis.

FIELD: peptides, pharmacy.

SUBSTANCE: invention relates to low-molecular derivatives of peptides that are able to act as inhibitors in interaction between laminine and nidogen (interactions laminine/nidogen). Also, invention relates to a method for their preparing, pharmaceutical composition prepared on thereof and their using for preparing pharmaceutical agents, and for identification of inhibitors in interaction laminine/nidogen.

EFFECT: valuable properties of peptides.

5 cl, 12 dwg

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