Oligopeptide having activity in the attachment, respectiveiy and detaching cells, and pharmaceutical composition
The invention relates to the field of biotechnology, specifically to peptides active in the attachment, respectiveiy and detaching cells, and can be used to study activity in the attachment of cells, mediated by different proteins of the extracellular matrix, to develop peptides, adhesion, and also for medicinal purposes. Oligopeptides stimulate the activity of attaching cells by interaction with integrin
3
1 as a functional cellular receptor, and include aspartic acid and isoleucine, are required for activity in the attaching and detaching of the cells. Pharmaceutical composition comprising an effective amount of oligopeptides, used for wound healing, tissue regeneration and inhibition of metastasis. The invention allows to obtain an Oligopeptide containing conservative amino acid sequence DI, necessary for the regulation of cell adhesion. 2 N. and 6 C.p. f-crystals, 15 ill.
The SCOPE of the INVENTION
This invention relates to peptides and their derivatives, active in the attachment, RSPM, which stimulate the activity of attaching cells by interaction with integrin31 as a functional cellular receptor, and include aspartic acid and isoleucine, are required for activity in the attaching and detaching of the cells.
The premise of the INVENTIONS
ig-h3 is a protein of the extracellular matrix, the expression of which is induced in various cell lines, including cells of human melanoma, epithelial mammalian cells, keratinocytes and fibroblasts easy, once the signal is active TGF-. First, geneig-h3 identified as a result of based on differential hybridization screening of a cDNA library derived from a cell line of human lung adenocarcinoma who were treated with TGF-. Geneig-h3 encodes a protein of 683 amino acids, which is highly conservative among different species. It contains N-terminal signal peptide for secretion and the motif Arg-Gly-Asp (RGD)-the end. The RGD motif, which modulates cell adhesion, found in many proteins vnekletocnam TGF-increases gene expressionig-h3 in different cell lines and gene is induced in different cell lines, the rate of proliferation are controlled by TGF-assume thatig-h3 is involved in mediating some of the signal transduction pathways of TGF-. On the contrary, it was reported that the expressionig-h3 is reduced in fibroblasts, kultiviruemykh areas of damage to the skin, the affected local hyperostosis when melorheostosis, in some tumor cells and stem cells treated with dexamethasone. Therefore,ig-h3 plays an important role in morphogenesis and interactions with cells and other proteins of the extracellular matrix in different tissues.
In addition, it is known thatig-h3 mediates the attachment and detachment of cells, performing the role of cell adhesion molecules. Found that the purified proteinig-h3 promotes attachment and raspletanie skin fibroblasts along with the inhibition of cell adhesion A, HeLa and Wi-38 in serum-free media. In particular, it is known that
ig-h3 causes the cells, especially fibroblasts, rasplachivatsa over the wound site and to stick with it. Soig-h3, a cell adhesion molecule, is induced by TGF-in different cell lines, plays a very important role in cell growth, cell differentiation, wound healing, morphogenesis and cell adhesion.
ig-h3 contains four repetition of 140 amino acids with internal homology. Duplicate inside the domains are highly conserved sequence found in secreted proteins or membrane proteins of different species, including mammals, insects, sea urchins, plants, yeast and bacteria. Such proteins, as periostin, fascicle I, HLC-2 sea urchin ITSELF algae and MRV mycobacteria are examples containing the conserved sequence. Homologous domain, conserved in these what Itami H1 and H2, each of which contains about 10 amino acids. Four domains of fas-1 detected inig-h3, periostin and fascicle I have two domains fas-1 HLC-2 and only one domain of the fas-1 in MRV. Although the functions of proteins is not exactly clarified, it is known that some of them act as cell adhesion molecules. For example, it is reported thatig-h3, periostin and fascicle I respectively mediate the adhesion of fibroblasts, osteoblasts and nerve cells. It is also reported that the algae is a cell adhesion molecule present in the germ algae Volvox.
First activityig-h3 in the attachment of cells found in fibroblasts of human skin, and then in chondrocytes, peritoneal fibroblasts and fibroblasts MRC5 human. Initially believed that the activityig-h3 in the attachment of cells, probably mediated by a C-terminal motif RGD. However, some studies revealed that the RGD motif is not required for stimulation of rasplastyvanija chondrocytes and that the Mature solubleig-h3, which deleterows the RGD motif in the processing carboxyl end sposo not essential to the mediation activityig-h3 in the attachment of cells. In addition, it was recently reported thatig-h3 promotes raspletanie fibroblasts via integrin11, whereas the RGD motifig-h3 is not necessary for indirectig-h3 rasplastyvanija cells. In addition, conservative peptides H1 and H2ig-h3 does not inhibit mediatedig-h3 cell adhesion, thus, conservative peptides do not affect the attachment of cell-mediatedig-h3. The results, taken together, suggest that the amino acids needed for activityig-h3 in the attachment of cells exist in other areas other than H1 and H2. Computer analysis of the homology domains fas-1 in other proteins, as well as repetitive domains of fas-1ig-h3 shows the presence of several highly conservative amino acid sequence, in addition to peptides H1 and H2, which indicates the possible participation of conservative amino acid sequence in AK is bretania intensive and comprehensive study of conservative motives, responsible for the activity of attaching and detaching cells, which led to this invention was the discovery of the fact that aspartic acid and isoleucine at positions near an area H2 in each 2nd and 4th fas-1 domainig-h3 highly conservative and identified as functionally necessary unit for mediation of cell adhesion through integrin31.
Therefore, the present invention relates to peptides and their derivatives, which contain conservative amino acid sequences required for activity in the attachment, respectiveiy and detaching cells.
Another object of the present invention are pharmaceutical compositions for use in wound healing, tissue regeneration and resistance to metastasis of malignant tumors.
According to one aspect of this invention relates to a peptide having activity in the attachment, respectiveiy and detaching cells containing the amino acid sequence represented by the following single-letter symbols: XXDIX, where X is any of the twenty conventional amino acids is worn to the pharmaceutical composition, containing the above-mentioned peptide or its derivative as a pharmaceutically active ingredient.
BRIEF DESCRIPTION of DRAWINGS
Fig.1A is a schematic diagram showing a recombinant protein derived from the domains of fas-1ig-h3.
Fig.1b is a photograph showing the results of SDS-page of recombinant proteins derived from the domains of fas-1ig-h3.
Fig.2 is a histogram showing the activity of recombinant proteins, obtained from the domains of the fas-1ig-h3 in the attachment of cells.
Fig.3 is a bar graph showing the inhibitory activity antiintegrin antibodies directed against the activity of recombinant proteins, obtained from the domains of the fas-1ig-h3 in the attachment of cells.
In Fig.4 shows the amino acid sequence of a different protein matrix containing domains fas-1.
Fig.5A is a schematic diagram showing the mutants with substitutions obtained from the 4th fas-1 domainig-h3.
Fig.5b is a photograph showing the results of electrophoresis in 15% polyacrylamide a histogram, showing activity in attaching cells of mutants with substitutions obtained from the 4th fas-1 domainig-h3.
In Fig.7 shows the amino acid sequences of synthetic peptides according to this invention.
Fig.8A is a bar graph showing the inhibitory activity of synthetic peptides directed against adhesion of cells and NSE.
Fig.8b is a bar graph showing the inhibition of cell adhesion and NSE proteinig-h3 D-II orig-h3 D-IV synthetic peptideig-h3.
Fig.9a is a curve showing the activity of the synthetic peptides according to the invention in the attachment of cells and NSE.
Fig.9b is a histogram showing inhibition antiintegrin antibodies of cell adhesion and NSE to synthetic peptides EPDIM and NKDIL.
Fig.10A is a histogram showing the effect of synthetic peptides according to the invention on cell adhesion and NSE to several proteins of the extracellular matrix.
Fig.10b is a curve showing the dose-dependent inhibition of cell adhesion and NSE to put on Britania
In this invention using the amino acid sequence ofig-h3, which, as you know, mediates the activity in the attachment of cells to obtain a peptide containing the conserved sequence required for activity in the attachment and respectiveiy cells, on the basis of the received data that onlyig-h3 containing 4 domain fas-1, and either the 2nd or 4th domain may mediate the activity in the attachment of cells.
In more detail, synthesized four short DNA fragment of the geneig-h3, namelyig-h3 D-I,ig-h3 D-II,ig-h3 D-III andig-h3 IV, which respectively encode the 1-St to 4-th internal repeat domains, as shown in Fig.1A. Quantitative measurement that shows the activity in the attachment and respectiveiy four cells resulting from recombinant proteins, demonstrates that either the 2nd or the 4th domain of four fas-1 domains can mediate activityig-h3 in attaching and respectiveiy cells, ka is rtraline and rasplastyvanija cells. On the contrary, the 1st domainig-h3 shows intermediate activity in attaching and respectiveiy cells, whereas in the case of the 3rd domain is not detected activity in the attachment of cells.
Also according to this invention identified integrin31 as a functional receptor for the 2nd and 4th domainsig-h3.
Using truncated proteins containing the 2nd and 4th domainsig-h3, each of which is active in the attachment, respectiveiy and detaching cells, the authors of this invention have investigated receptorsig-h3. Activity in the attachment of cell-mediated 2-m or 4-m domains fas-1, almost completely inhibited by antibodies against subunits3 and1 integrin, as shown in Fig.3. The obtained results indicate that both domains 2 and 4, associated with the functional receptor of the integrin31, Poreba activity in the attachment of cells, and have amino acids that are required in order to mediate the activity of the active amino acid sequence Asp-Ile, required for activityig-h3 in the attachment, respectiveiy and detaching cells, and therefore show the same activity as the 2nd and 4th domains.
To detect amino acid sequence responsible for cell adhesion, in the 2nd and 4th fas-1 domainig-h3, which independently active in the attachment, respectiveiy and detaching cells, performed the alignment of amino acid sequences not only between internal duplicate fas-1 domainsig-h3, but also between other proteins containing domains of the fas-1. After that, the authors of this invention have found that two amino acids, aspartic acid and isoleucine near an area H2 are highly conservative among different proteins, as shown in Fig.4.
We confirm the necessity of the amino acid sequence of aspartic acid and isoleucine for activity in the attachment of cells. This made the mutation truncated protein containing the 4th fas-1 domainig-h3, replacement of Proline, aspartic acid and isoleucine serine by alanine and serine, respectively, as of pocetak mutant proteins which were replaced with aspartic acid and isoleucine, which confirms that the amino acid sequence of aspartic acid and isoleucine is very important in mediating activityig-h3 in the attachment of cells.
In addition, the two amino acids - aspartic acid and isoleucine, are conservative in the 2nd and 4th fas-1 domainig-h3, which have high activity in the attachment of cells, whereas in 1-m fas-1 domain 1, which shows an intermediate activity in the attachment of cells, only conservative aspartic acid. As for the 3rd fas-1 domain, which is inactive in the attachment of cells, it lacks two amino acids. The resulting facts are further evidence showing that aspartic acid and isoleucine is necessary in order to mediate the activity of attaching and respectiveiy cells.
Affirming the need aspartic acid and isoleucine for activity in the attachment of cells, synthesized three peptide containing two amino acids of fas-1 domains I, II and IVig-h3, respectively. These peptides have frowny so, so they had KADHH (a/K 219-223) SEQ ID NO. 1, NKDIL (a/K 354-358) SEQ ID NO. 2 and EPDIM (a/K 615-619) SEQ ID NO. 3, which is shown in Fig.7.
That needless to say, these synthetic peptides derived from fas-1 domainsig-h3, was investigated in relation to activity in the attachment and respectiveiy cells. Estimated synthetic peptide NKDIL obtained from the 2nd fas-1 domain, and a synthetic peptide EPDIM obtained from the 4th fas-1 domain, to show excellent activity in detaching cells with significant suppression of activity in the attachment of cells, as seen in Fig.8A. On the other hand, relatively low impact on the detachment of cells was observed in the case of synthetic peptide KADHH obtained from 1 fas-1 domain, due to the weak suppression of cell adhesion, as well as a control peptide DEMPI, does not exhibit effects due to inactivity in the adhesion of cells. Results suppression directed against the activity in the attachment of cells obtained in the case of the 2nd and 4th fas-1 domains as substrates, were almost the same as the results obtained when using as substrateig-h3, as shown in Fig.8b.
As shown in Fig.9 the go, what integrin31, known as surface receptorig-h3, acts as the receptor for mediated synthetic peptides EPDIM and NKDIL adhesion of cells, as seen in Fig.9b. For these reasons NKDIL and EPDIM apparently compete with specific molecules that interact with31, as shown in Fig.10A.
As described above, the present invention presents peptides, NKDIL and EPDIM that contain aspartic acid and isoleucine, are required for activity in the attaching and detaching of the cells, and which are sufficient to induce cell adhesion through integrin31, which is the functional receptor forig-h3. These peptide sequences derived from conserved sequence fas-1 domains II and IV, which independently induces cell adhesion. The peptides according to this invention can be successfully used to study activity in the attachment of cells, mediated by different proteins of the extracellular matrix, and the detaching of the cells.
According to further aspect of the present invention presents pharmaceutical compositions containing the peptides active in the attaching and detaching of the cells, or their derivatives as effective ingredients, giving the ability to heal wounds and to carry out the regeneration of tissues and resistance to metastasis of malignant tumors.
The peptides or their derivatives, administered by oral or parenteral routes, can be used in conventional dosage forms. That is, the peptides or their derivatives can be prepared in the form of various dosage forms for parenteral administration. For the preparation of compositions can be used pharmaceutically acceptable diluents, excipients and/or carriers, including excipients, thickeners, binders, moisturizing agents, disintegrators, surfactants, etc. Dosage forms for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized tools, suppositories, etc. For the preparation of solutions in non-aqueous solvents, suspensions, you can use vegetable oil such as propylene glycol and Poliany witepsol, macrogol, tween 61, cacao butter, lauric acid and glycoregulation.
In addition, peptides and their derivatives can be used in combination with pharmaceutically acceptable carriers, such as biologically active salt or organic solvents. Also to improve the stability or absorption of peptides can be applied carbohydrates, such as glucose, sucrose and dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, proteins with low molecular weight or other stabilizers.
The total effective amount of the peptides can be introduced in the form of a metered bolus form or you can enter through infusion in one dose in the case where it is desired relatively short period of introduction. Alternatively, when the introduction of peptides is possible to observe how multiple doses according to the Protocol of fractional treatment. Depending on the age, body conditions and body weight of patients, and pathways of introduction and the number of treatments pharmaceutically effective dose of the peptides according to this invention can vary, and it can easily determine the experts in this field.
As pharmaceutical compositions containing as is.
EXAMPLES
To better understand this invention is possible in light of the following examples, which are provided for illustration and are not intended to limit the present invention.
EXAMPLE 1: creating a recombinant protein fas-1 domainsig-h3 and analysis of activity in the attachment of cells
To identify amino acidsig-h3, required for adhesion of cells was determined by the ability of each repeated four fas-1 domains to mediate the adhesion of cells toig-h3. To this end, to analyze the activity of the attached cells were created four recombinant protein, which contained four repetitive domain of the fas-1.
1-1: preparation of recombinant proteins fas-1ig-h3, coding each of the four repeated domains
Through PCR has created four DNA fragment encoding the 1-St to 4-th domains, corresponding to bases 133 through 236, 242 at 372, 373 on 501 and 502 in 632 geneig-h3, respectively, and cloned in pET-29(Novagen) to construct expressing vectors for domains fas-1, namedig-h3 D-1,. Strain BS21(DE3) E. coli transformed each recombinant expressing vector and cultured in media LB containing 50 µg/ml kanamycin. Recombinant proteins<img src=)
In SDS-page of recombinant proteinig-h3 D-I were identified in the area of 14.4 KD, whereas all other recombinant proteinsig-h3 D-II,ig-h3 D-III andig-h3 D-IV, were identified in the area of 21.5 KD, as shown in Fig.1b.
1-2: analysis of the activity of recombinant proteins fas-1 domains
ig-h3 was given the opportunity to stick to the bottom 96-well plates for micrometrology (Falcon) by incubation at 37°C for 1 hour and blocked PBS containing 0.2% BSA. Cells NSE suspended in the medium for culturing at a density of 2×105cells/ml In each well of tablets coated with recombinant proteins, was added 0.1 ml of cell suspension. After incubation at 37°C for 1 hour unattached cells were removed by PBS washing. Attached cells were incubated for 1 hour at 37°C in 50 mm citrate buffer, pH 5.0, containing 3.75 mm para-NITROPHENOL-N-acetyl-1--D-glucosaminide as substrate hexosaminidase, and 0.25% Triton X-100, after which was added 50 mm glycine buffer, pH of 10.4, containing 5 mm EDTA to block the enzymatic activity. The density measurement was carried out at 405 nm in the reader for microplates Multiscan MCC/340. Data optical density shown in Fig.2.
As can be seen in the histogram of Fig.2, the cell of NSE was active in the attachment and respectiveiy cells, Crewplanet, coated with recombinant protein of the 2nd fas-1 domain (igh3-D-II) and the recombinant protein of the 4th fas-1 domain (igh3-D-IV), with weak activity was manifested in the tablet coated with recombinant protein 1 fas-1 domain (igh3-D-I). On the other hand, almost no activity in the tablet coated with recombinant protein fas-1 domain III (igh3-D-III).
Either the 2nd or the 4th domain of the fas-1 is sufficient to mediate adhesion and raspletanie cells, suggesting that amino acids necessary for adhesion and rasplastyvanija cells, are present in each of the two domains.
1-3: Identification of receptors for protein fas-1 domainsig-h3
Using recombinant proteins of the 2nd and 4th domainsig-h3, each of which was active in the attachment and respectiveiy cells, investigated receptorsig-h3. From this point of view investigated the effect of blocking the functioning of monoclonal antibodies against different subunits of the integrin in cell adhesion and NSE to the surface, covered withig-h3.
Sublattice is of monoclonal antibodies (5 μg/ml) against different types of integrins at 37°C for 30 minutes The pre-incubated cells was transferred into tablets, pre-coated proteinsig-h3, and then incubated at 37°C for 1 hour, and then conducted a quantitative analysis with the substrate hexosaminidase as described in example 1-2. Data of the quantitative analysis shown in Fig.3, where the values are expressed in percentage of the number of cells adhering in the absence of monoclonal antibodies.
As can be seen in Fig.3, adhesion of cells, mediated by the 2nd or the 4th fas-1 domains, almost completely inhibited by both antibodies against integrin subunits3 and1. The results obtained indicate that both fas-1 domain, 2-nd and 4-th, contact integrin31, Poreba activity in the attachment of cells, and have the amino acids necessary for mediation.
EXAMPLE 2: Amino acids, aspartic acid and isoleucine, are required for activityig-h3 in attaching and respectiveiy cells
2-1: Alignment of amino acid sequences of different proteins containing domains of the fas-1
To identifizierung amino acid sequences were subjected not only to the internal repetitive fas-1 domainsig-h3, but also other proteins containing fas-1 domains, each of which has activity in attaching and respectiveiy cells. In the result, it was found that two amino acids, namely aspartic acid and isoleucine, are highly conservative in the site near an area H2 of each domain of the fas-1 among the different substrate proteins, as shown in Fig.4. In General, and aspartic acid and isoleucine are conservative in different domains of the fas-1, including the 2nd and 4th domainsig-h3, whereas in 1-m domain of the fas-1 only conservative aspartic acid. As for the 3rd domain of the fas-1, it does not contain any of the two amino acids.
2-2: development of recombinant proteins containing mutational substitutions in the 4th fas-1 domainig-h3
In order to investigate the necessity of aspartic acid and isoleucine for activityig-h3 in the adhesion of cells, created mutants of recombinant proteinig-h3 D-IV, obtained from 4-th domainig-h3, in which Proline at position 616, aspartic acid at position 617 and isoleucine at position 618 replaced by serine, alanine and serine, respectively. Received>/img>igh3 D-IV-PaIigh3 D-IV PDs andigh3 D-IV-sas (Fig.5A). In SDS-page all mutant recombinant proteins were detected in the same position asigh3 D-IV (Fig.5b).
2-3: Analysis of activity in the attachment of cells of recombinant proteins containing mutational substitutions in the 4th fas-1 domainig-h3
A study of activity in the attachment of cells of mutant proteins in which Pro616, Asp617 and Ile618igh3 D-IV in combination were replaced, respectively, Ser, Ala and Ser, that is,igh3 D-IV-sDI,igh3 D-IV-PaIigh3 D-IV PDs andigh3 D-IV-sas. With this purpose, cells and NSE were incubated at 37°C for 1 hour in the wells of tablets, coated mutant proteins, then the attached cells were exposed to the effect of substrate hexosaminidase as described in example 1-2. The results are presented in Fig.6.
As can be seen in Fig.6, the mutant protein containing Ala instead Asp617 called D617A (igh3 D-IV-PaI), and the mutant protein containing Ser instead Ile618 called I618S (igh3 D-IV PDs), to a large degree the 616S (igh3 D-IV-sDI), has been active in the attachment of cells, comparable to the activity of control wild-type. As for the mutant protein, in which the mutant were three amino acids called P616S/D617A/I618S (igh3 D-IV-sas), its activity in the attachment of cells was also much lower than in the control.
The loss of activity in the attachment of cell-mediated 1-m domain of the fas-1, 1-m domain of the fas-1, a mutant in position Asp617 or Ile618 suggests that the aspartic acid at position 617 and isoleucine at position 618 are very important in mediating activityig-h3 in the attachment of cells.
The results obtained are consistent with the results of example 1. Analyzing the results of example 1, we can say that aspartic acid and isoleucine, which are identified as necessary for mediation activity in the attachment of cells, conservative as in the 2-m and 4-m domains of the fas-1 active in the attachment of cells, whereas in 1-m domain of the fas-1, which shows weak activity in the attachment of cells, only conservative aspartic acid. On the other hand, the 3rd domain that does not have activity in the attachment of cells, not coder very important for mediating activityig-h3 in attaching and respectiveiy cells.
EXAMPLE 3: Synthetic peptides based on theig-h3, with activity in the attachment, respectiveiy and detaching cells
Affirming the need aspartic acid and isoleucine for activity in the attachment of cells, synthesized peptides containing two amino acids, and analyzed the activity in the attaching and detaching of the cells.
3-1: Synthesis of proteins derived from fas-1 domainig-h3
Synthesized peptides derived from the 1st, 2nd and 4th fas-1 domainsig-h3, and the control peptide using an automatic synthesizer of various peptides (PE/ABD 433) standard methods of solid-phase synthesis, peptides were isolated and purified by reversed-phase high-performance liquid chromatography. The synthesized peptides were designed so that they had KADHH (a/K 219-223) SEQ ID NO. 1, NKDIL (a/K 354-358) SEQ ID NO. 2 and EPDIM (a/K 615-619) SEQ ID NO. 3, corresponding to the conservative sequences of the 1st, 2nd and 4th fas-1 domainsig-h3, respectively, and the control peptide DEMPI had the same amino acid composition as the peptide EPDIM, but modified poalini and detaching cells
The effect of peptides containing the conserved sequence of fas-1 domainsig-h3, mediatedig-h3 cell adhesion was investigated using cells and NSE.
The cells were pre-incubated for 30 minutes in media containing 100 μm each of the four synthetic peptides, or without peptides and transferred into plastic cups for cultivation, covered with 10 μg/ml BSA or 10 µg/mlig-h3. Attached cells were subjected to quantitative analysis to hexosaminidase as in example 1-2. The results are presented in Fig.8A.
As shown in Fig.8A, synthetic peptides NKDIL and EPDIM, each of which contains conservative residues aspartic acid-isoleucine, significantly blocked cell adhesion and NSE toig-h3, showing great activity in detaching cells, whereas synthetic peptide KADHH containing only conservative aspartic acid, weakly inhibited cell adhesion. On the other hand, the control peptide DEMPI that do not contain any of conservative amino acid residues, did not affect cell adhesion and consequently had low is>/img>igh3 D-II andigh3 D-IV also investigated in the presence or absence of synthetic peptides. With this purpose, cells, there is pre-incubated for 30 minutes in media containing 100 μm each of the four synthetic peptides, or without peptides and then transferred into plastic cups for cultivation, covered with 10 µg/mlig-h3 D-II or D-IV. After incubation, the attached cells were quantitatively analyzed in relation to hexosaminidase as in example 1-2. The results are presented in Fig.8b.
As shown in Fig.8b, the blocking effect of synthetic peptides obtained in the case when the substrates used 2nd and 4th domains fas-1, was similar to the effect obtained in the case when the substrate usedig-h3.
3-3: Cell adhesion of cells and NSE to surfaces coated with synthetic peptides
The experiment was performed in order to determine whether each of the synthetic peptides to mediate cell adhesion. Hole tablets were covered with each peptide at a concentration of 0, 20, 40, 60, 80, 100 and 120 μm and then incubated with cells and NSE at 37°C for 1 hour. Attached CA.
Found that synthetic peptides NKDIL and EPDIM, each of which contains as aspartic acid and isoleucine, mediate cell adhesion dose-dependent manner. Synthetic peptide KADHH also had the ability to mediate cell adhesion dose-dependent manner, but its activity was lower compared to NKDIL and EPDIM. As expected, the control peptide that does not contain either one of the two amino acids, was inactive in the adhesion of cells.
3-4: Identification of receptors for mediating activity of synthetic peptides in the attachment, respectiveiy and detaching cells
Conducted experiments on blocking operation, using monoclonal antibodies against different subunits of the integrin to explore, involved whether the integrin31, known as surface receptorig-h3, cell adhesion, mediated by synthetic peptides EPDIM and NKDIL. The NSE pre-incubated at 37°C for 30 min in the presence of each of the monoclonal antibodies (5 μg/ml) against different types of integrins. The pre-incubated cells was transferred into tablets, pre-coated with 100 µm Ecomodo substrate hexosaminidase, as in example 1-2. Quantitative results are shown in Fig.9b.
As can be seen in Fig.3, adhesion of cells, mediated by the 2nd or the 4th fas-1 domains, almost completely inhibited by both antibodies against integrin subunits3 and1. The results obtained indicate that both fas-1 domain, 2-nd and 4-th associated with integrin31, Poreba cell adhesion.
3-5: Effect of synthetic peptides on cell adhesion to different peptides of the extracellular matrix
To determine whether the inhibitory activity of synthetic peptides EPDIM and NKDIL directed against the adhesion of cells specific toig-h3, investigated the effect of synthetic peptides on cell adhesion to different proteins of the extracellular matrix. Cells, there is pre-incubated in media with addition of 100 μm each of synthetic peptides EPDIM or NKDIL, or without them, and then sprayed on the tablets coated with different proteins of the extracellular matrix, includingig-h3, fibronectin, laminin, vitronectin, collagen type I and type II. After 1 hour incubation attached coli cells is shown in Fig.10A, synthetic peptides NKDIL and EPDIM effectively blocked cell adhesion not only toig-h3, but also to laminin, showing great activity in detaching cells. However, they are poorly inhibited cell adhesion to fibronectin and generally had no effect on cell adhesion to collagen type I and type II and vitronectin. Thus, taken together the results indicate that synthetic peptides NKDIL and EPDIM compete with specific molecules that interact with integrin31.
The adhesion of cells toig-h3, laminin and fibronectin, which are detected as being blocked by synthetic peptides NKDIL and EPDIM, investigated at various concentrations EPDIM. Cells and NSE were incubated in media containing synthetic peptide in concentrations EPDIM 0, 200, 400, 600, 800, 1000 and 1200 μm, and inflicted on tablets covered withig-h3, fibronectin and laminin. After incubation for 1 hour attached cells was quantitatively analyzed in relation to hexosaminidase as in example 1-2. The results are shown in Fig.10b.
As can be seen on the curves, the adhesion of cells toig-h3, fibronectine action EPDIM on the adhesion of cells toig-h3, fibronectin and laminin depends on the dose.
In this invention, as described above, the obtained synthetic peptides NKDIL and EPDIM, which both contain aspartic acid and isoleucine required in the conservative motives for activity in the attachment and respectiveiy cells. The peptides contain the conserved sequence of the 2nd and 4th fas-1 domainsig-h3, it was discovered that each domain has been active in the attachment of cells, and they induce cell adhesion through integrin31, known as a functional cellular receptor. In the case of different extracellular matrix proteins, which are known to be associated with integrin31, such as thrombospondin, laminin, collagen type IV, and so on, still there was not a characteristic sequences in their active sites with homology nor typical conservative binding motifs for integrin31. However, after receiving evidence that synthetic peptides NKDIL and EPDIM, each of which contains both conservative="0">31, in order to mediate the activity in the attachment of cells according to this invention conservative amino acid aspartic acid-isoleucine identify as typical conservative binding motif for integrin31. Therefore, the peptides according to this invention can be successfully used to study activity in the attachment of cells, mediated by different proteins of the extracellular matrix, includingig-h3, and for the development of peptides that stimulate the attachment, raspletanie and unpinning cells.
INDUSTRIAL APPLICABILITY
According to this invention found that of the four fas-1 domainsig-h3, which is a cell adhesion molecule, 2-nd and 4-th domains are active in the attaching and detaching of the cells, and each of them contains a conservative sequence aspartic acid-isoleucine, necessary for activity in the attaching and detaching of the cells. After receiving these results, created peptides NKDIL and EPDIM that contain functional amino acid residues and indusiry according to this invention can be successfully used to study activity in the attachment of cells, mediated by different proteins of the extracellular matrix, includingig-h3, and for the development of peptides that stimulate the attachment, raspletanie and unpinning cells.
The invention is described in an illustrative manner, and it should be understood that we mean that the terminology used is more in the nature of description and not of limitation. There are numerous modifications and variations of the present invention in light of the above instructions. Therefore, it should be understood that in the framework of the attached claims practice of the invention may be carried out otherwise than specifically described method.
Claims
1. Oligopeptide having activity in the attachment, respectiveiy and detaching cells containing the amino acid sequence represented XXDIX, where X can be any of the twenty standard amino acids.
2. Oligopeptide under item 1, consisting essentially of aspartic acid and isoleucine as a conservative sequence.
3. Oligopeptide under item 2, where the amino acid sequence represented NKDIL SEQ ID NO: 2.
4. Oligopeptide on Yunosti attaching and respectiveiy cells through integrin31.
6. Oligopeptide under item 1, showing inhibitory activity against adhesion of cells toig-h3, fibronectin or laminin.
7. Pharmaceutical composition having activity in the attachment, respectiveiy and detaching cells, characterized in that it contains an effective amount of oligopeptides under item 1 and pharmaceutically acceptable additives.
8. The pharmaceutical composition according to p. 7, therapeutically effective for wound healing, tissue regeneration or resistance to metastasis of malignant tumors.
and the polymer containing lactide links, glycolide links and links tartaric acids - which are found in the polymer at the next sootnoshenii: lactide units constitute from about 71% to about 73%, glycolide links from about 26% to about 28%; and the parts of tartaric acid from about 1% to 3%, and the amino group of the compound (a) relate ionic bond with the carboxyl groups of the acid units of the polymer; the particles of compound I, an average size of from about 10 microns to about 100 microns; pharmaceutical composition with delayed release and two methods of treatment of various diseases, including the introduction to the patient an effective amount of compound A, or microparticles
in which a represents acetyl or 3-(4-forfinal)propionyloxy group Xxx1mean D-Nal(1) or D-Nal(2), Xxx2-Xxx3mean D-Cpa-D-Pal(3) or a simple link, Xxx4means Ser, Xxx5means N-Me-Tyr, Xxx6mean D-Hci or a residue of D-amino acids of General formula (II)
where n means the number 3 or 4, a R1means a group of the General formula III
where R denotes an integer from 1 to 4, R2means hydrogen or alkyl group, and R3means unsubstituted or substituted aryl group or heteroaryl group, or R1mean 3-amino-1,2,4-triazole-5-carbonyl group,
Xxx7means Leu or Nle, Xxx8means Arg or Lys(iPr), Xxx9means Pro and Xxx10means A1A or Sar, and their salts with pharmaceutically acceptable acids: process for the preparation of these compounds, pharmaceutical compositions having the properties of an LHRH antagonist, comprising as an active narushenie compounds according to the invention has a high solubility in water
domain antigen In human leukocyte (HLA-B) 75-84, (b) a naturally occurring sequence of the transmembrane sequence-chain T-cell receptor of human rights and (in) consistency, which is the mutant with respect to either (a) or (b) having no more than two mutations as an active part of the above oligopeptides; the method of suppressing the activation limfozitah cells, the method of transplantation of donor organs or mammalian cells, methods of inhibiting protein synthesis of inflammatory cytokines by cells capable of producing the specified protein inflammatory cytokine, the method of suppressing an inflammatory response in a mammal, the method of modulation of the activity hemade the
where R1, R2and R3are H, HE or och3group and R4represents the following groups:
The proposed pharmaceutical composition having antitumor activity, containing at least one derivative of 5-imino-13-deoxy of anthracycline
