Rabbit anticavity, inhibiting the transport of cationic amino acids, and containing pharmaceutical composition


(57) Abstract:

The invention relates to pharmacology, and in particular to means, inhibiting the transport of cationic amino acids, and reveals rabbit anticigarette induced against T-cell protein encoded by the genome of the IRU-2, and pharmaceutical composition comprising the specified anticigarette. The invention provides inhibition of transport of cationic amino acids. 2 c.p. f-crystals, 22 ill., 5 table.

Prior art

The technical field to which the invention relates

The invention relates mainly to new DNA sequences encoding vectors of cationic amino acids. More specifically, the present invention relates to new ways of using these DNA sequences and antibodies against the expressed proteins for the treatment of pathophysiological disorders.

Description prototypes

the+Transportation system facilitates the transport of cationic amino acids (AA), such as arginine, lysine and ornithine, sodium-independent way. Recently it was reported Dr. J. Cunningham (Harvard medical school) on the allocation of cDNA-clone vector cationic AA. Other cDNA clones isolated and proteins are directly related to the transport of AA. These proteins are referred to as ancillary proteins or protein-activators.

Gene IRU-2 was originally called Thea-genome. However, in the following called the carrier of murine cationic amino acids (murino Cationic amino acid fransporter) (mCAT-2) through the functions described in this application.

Arginine, which is necessary for synthesis of proteins, plays a major role in the biosynthesis of other AA and is the immediate precursor of urea in the urea cycle. Arginine is necessary for the synthesis of high-energy phosphagen, creatine phosphate, which is carried out by transfer of donor-amedieval group to glycine in the first stage of creatine synthesis. The liver is not a pure supplier of arginine due to the high level of arginase. Arginine exchange between the kidneys and the circulatory system needs and transport mechanisms for input and output of arginine in the glomerular filtration. Therefore, every organ in the body, in addition to liver and kidneys, gets arginine from plasma by transport mechanisms. In contrast, lysine, which is an essential amino acid that must be ingested with food. This amino acid is not synthesized by mammals which.

Arginine has a strong stimulating effect on the endocrine glands. Intravenous or oral administration of arginine adult stimulates the secretion geospizinae growth hormone, prolactin and insulin. In addition, arginine affects the immune system of the body regardless polienovogo synthesis.

Arginine is the sole precursor responsible for the synthesis of nitric oxide (NO). NO is the most powerful of the known vetmousetrap, and is a necessary element for the implementation of the normal functions of macrophages and T-cells. From nitric oxide dependent cytotoxic activity of macrophages and the production of NO in vascular endothelium regulates blood pressure, and, in addition, NO is a neuromodulator. Like all free radicals, NO is highly reactive, and therefore highly unstable compound, which is rapidly converted to nitrate or nitrite. Production of NO is partially regulated by IL-2, TNF-alpha and TNF-gamma.

In the previous studies there are no descriptions effective methods of regulation of the production of nitric oxide. Therefore, the urgent need for the development of such methods was preposal the implementation the present invention relates to the recombinant polypeptide, containing the amino acid sequence of T-cell protein encoded by the genome of 20.5.

In another embodiment, the present invention relates to anticigarette produced against two forms of mCAT-2 protein.

In yet another embodiment, the present invention relates to pharmaceutical compositions containing anticigarette produced against mCAT-2 proteins.

In another embodiment, the present invention relates to a method for inhibiting the transport of cationic amino acids, namely, that the person or other animal is administered a pharmacologically effective dose of the pharmaceutical composition of the present invention.

In the following embodiment, the present invention relates to a method of inhibiting the production of nitric oxide, which consists in the introduction of the animal a pharmacologically effective dose of the pharmaceutical composition of the present invention.

In yet another embodiment, the present invention relates to a method for the treatment of pathophysiological conditions in the animal associated with produced undesirable levels of nitric oxide, and this method provides for the introduction of this animal a pharmacologically effective until the global features of the present invention are evident from the following description of the preferred variants of its implementation, illustrated below the images.

Brief description of drawings

The following drawings are presented to illustrate the various options and ways to implement the present invention. For clarity and illustration purposes, some of these drawings are given in schematic form, and some fragments are shown in a larger view.

Figure 1 illustrates the expression of 5 different SL12.4-specific cDNA clone, which was determined using Northern blot analysis.

Figure 2 illustrates a southern blot-hybridization of different SL12.4-specific cDNA clones.

Figure 3 shows the DNA and deduced protein sequence for the cDNA clone 20.5 (mGAT-2).

Figure 4 illustrates the expression of mRNA of the gene IRU-2 (denoted earlier Tea) cell lines SL12.3 and SL12.4.

Figure 5 illustrates the expression of total cellular, cytoplasmic and nuclear mRNA pvsat-2 cell lines SL12.4.

Figure 6 illustrates the expression of mRNA IRU-2 series murine cell lines.

Figure 7 illustrates the expression of mRNA of the gene IRU-2 in these normal tissues of Balb/c mice. CALT means lymphoid t is 2 in activated splenocytes.

Figure 9 illustrates the kinetics of induction of gene expression Tea or IRU-2 in activated splenocytes. Control sensing indicates the relative amount of mRNA loaded in each lane.

Figure 10 illustrates a comparative analysis of the primary cDNA sequences 20.5 or IRU-2 and ERR.

Figure 11 (A-D) compares the primary sequence 20.5 cDNA (top) and cDNA KRR (bottom).

Figure 12 illustrates a comparative analysis of the predicted protein sequence encoded mGAT-2 gene, and sequence ecotropica retroviral receptor.

Figure 13 (a-b) shows the comparison of the predicted sequence mGAT-2 protein sequence ecotropica retroviral receptor.

Figure 14 shows southern analysis of DNA from different species of animals.

Figure 15 shows southern analysis of recombinant inbred DNA in position rnCAT-2 gene on chromosome 8.

Figure 16 illustrates the substrate specificity of the IRU-2-mediated amino acid transport in Xenopus oocytes. The transport was assessed by measuring the internal currents recorded in response the EN 30 or 75 ng mCAT-2-crnc or DEPC-water. The values obtained represent the average changes of the current average square Osh. at a fixed potential of about 60 mV.

Figure 17 illustrates steric specificity and degree of dependence mCAT-2-mediated transport from the presence of sodium. Steric specificity was assessed in oocytes injected with gpsat-2-crnc (30 or 75 ng) treated with 1.0 mm D - and L-isoforms of arginine, lysine or ornithine. The data obtained represent the average changes of the current average square Osh. for 5-6 different oocytes (P<0,0001 for arginine, lysine and ornithine).

Figure 18 illustrates the sodium-independent transport in oocytes injected with gpsat-2-crnc. The DNA sequence 20.5 designated as SEQ ID 1, and the predicted amino acid sequence designated as SEQ ID 2. Sodium-dependent transport in oocytes injected with mCAT-2-cDNA (30 or 75 ng) was evaluated by measuring the current in the presence and in the absence of sodium. The NaCl was replaced with equimolar amounts of holdingarea. Then using 5-6 oocytes constructed a chart of the average change current cf. square Osh. Fixed potential was ~60 mV. Concentrations of arginine (P= 0,5248), lysine (P<0,4326), ornithine (0,9191) was 1.0 mm, and homoserine the bathroom rnCAT-2-crnc were perfusion at concentrations of arginine, lysine, ornithine and histidine from 0.01 mm to 30.0 mm. The data obtained represent the average changes of the current average square Osh. for 3-7 oocytes, which were pre-injected 50-200 ng gpsat-2-crnc. Inset: linear regression data saturation corresponds to a sigmoidal curve using GraphPad program InPlot 4.03. Using the linear regression values were determined Imaxand Km. The DNA sequence 20.5 designated as SEQ ID 1, and the DNA sequence ERR designated as SEQ ID 3.

Figure 20 illustrates Northern analysis IRU-2 - and mCAT-1 transcripts in normal tissues. The DNA sequence 20.5 designated as SEQ ID 8, and the DNA sequence ERR designated as SEQ ID 9. Total RNA was obtained from tissues of adult animals and cell lymphoma SL 12.4. The predicted amino acid sequence of the protein IRU-2 was identified as SEQ ID 2, and the amino acid sequence ERR designated as SEQ ID 4. Northern blots were obtained in duplicate and probed for the expression or IRU-2, or IRU-1. Cyclophilin probe was used to control for RNA loading. To illustrate the size of the transcript on the right also shows the mCAT-2-probed RNA skeletal muscle, liver and SL 12.4 p is Fig.21 And shows duplicate Northern blots total RNA from cells T-lymphoma SL 12.4 and two lines Hepatol HTI (NTS) and NT2 (HEPA SS), which were probed the SAT-1 and IRU-2, as indicated in Fig. 21A. These blots were stained with methylene blue to illustrate the number of 18S - and 28S-rRNA; pictures placed below the autoradiograms. Figure 21B shows total RNA tissue control liver, regenerating liver and liver lonaprisan animals. To compare the amount of RNA present in each filter, these RNAS were probed IRU-2 and cyclophilin (WM). The predicted amino acid sequence of the protein IRU-2 was identified as SEQ ID 2, and the amino acid sequence ERR designated as SEQ ID 4. Each lane represents RNA from an individual animal. Figure S shows a Northern blot of RNA (indicating tissue), which was probed using 132 p. O. - mCAT-2-fragment, shown on the right side of Fig.S to illustrate the differences transcipt from IRU-2A.

Figure 22 schematically shows the structure predicted mCAT-2 protein.

Detailed description of the invention

The present invention relates to new DNA sequences and to anticigarette, which is associated with proteins encoded by these DNA sequences. In another embodiment, the present invention relates to a cDNA clone (splenocyte, activated by mitogen Sopa for T cells.

DNA for the indicated T-cell protein of the present invention can be obtained from a mammal of any kind. Thus it is only necessary that the genetic sequence for the T-cell protein (TCP) expressives in prokaryotic or eukaryotic organism. Preferably, if the DNA expressing the T-cell protein (TCP), is derived from mouse. Particularly preferred is a DNA sequence encoding a T-cell protein that is capable of immunological cross-reactions between different species of animals (e.g. mouse, rabbit, sea lion or a person).

Recombinant T-cell protein can contain a complete amino acid sequence of TCP-protein or it may contain only specific determinants. Animal immunized with T-cell recombinant protein, will produce antibodies that bind to epitopes present on the recombinant or natural polypeptides. Thus, can be carried out industrial production of T-cell recombinant proteins.

Thanks to the present invention can be obtained anticancerogenic proteins of the present invention and as an inhibitor of binding of natural ligands to transmembrane T-cell proteins of the present invention, and also as a medicinal product or system designed shipping labels to the target.

The present invention relates to the recombinant polypeptide comprising the amino acid sequence of T-cell protein encoded gene 20.5, and under the new designation genome IRU-2.

In addition, the present invention relates to polyclonal antibodies produced against the protein, stated in paragraph 1 of the claims, as well as to pharmaceutical compositions containing anticigarette stated in paragraph 2 of the claims.

The methods of the present invention can be applied to any animal. However, it is most preferable if new compositions used in the methods of the present invention, will be introduced to the man.

In General, new antitelomerase pharmaceutical compositions used in the methods of the present invention, can be entered in all doses, inhibiting the production of nitric oxide in the animal. Any expert working in the field of pharmacology and pharmacokinetics, can easily determine the appropriate dose of a new pharmaceutical compositions of the present invention.

Basically, the FPIC of the state, associated with production in the body unwanted levels of nitric oxide. The method of the present invention is preferably used for the treatment of diseases such as Kaposi's sarcoma, cerebral malaria syndrome capillary permeability and autoimmune diseases. Typical autoimmune diseases are systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis.

Dose compositions of the present invention, the input according to the method described in this application depends on the age and weight of the patient, from the way the competitive treatment (if possible) and on the nature of the pathophysiological condition. The effective composition of the present invention can be manufactured in the form of capsules, tablets, artificial lipid vesicles to encapsulate, liquid solutions, suspensions, or elixirs for oral administration, or sterile liquid forms such as solutions, suspensions or emulsions. While it is preferable to use an inert carrier, such as physiological or potato-buffer solution, or any medium in which the compounds used in the methods of the present invention, possess the appropriate solution is farmacevtichesky acceptable carrier. As the pharmaceutically acceptable carrier may be any solvent which is compatible with the antibody of the present invention and which is non-toxic in the quantities entered patients. Pharmacological dose of new compounds containing the antibody used in the methods of the present invention, corresponds to the quantity of this compound, the inhibitory production of nitric oxide.

The present invention also relates to a method for inhibiting the transport of cationic amino acids, including arginine, which consists in the fact that a person or other animal is administered a pharmacologically effective dose of the above pharmaceutical compositions. In addition, the present invention relates to a method of inhibiting the production of nitric oxide, which consists in the fact that the animal is administered a pharmacologically effective dose of the above pharmaceutical compositions. There are three types of vectors carrying out bidirectional cation transport. Any of these carriers can be selectively locked to the implementation of other transport carriers. Even if one of these types of carriers is dominant in relation to the substrate on the TEI, encoding the protein of the present invention.

For a more complete understanding of the present invention the following are specific examples. These examples are given only for illustrative purposes and should not be construed as a limitation of the present invention.

Example 1. Isolation, characterization and cultivation of cells

Isolation, characterization and culturing conditions cell lines T-lindome, SL12.1, SL12.3 and SL12.4 and formed by somatic cell hybrids described in detail in: Hays, etc.. Int.,J. Cancer. 38: 597-601 (1986); MacLeod and others, Cancer Research 44: 1784-1790 (1984) and MacLeod and others, Nat. Cancer Inst 74: 875-882 (1985).

The phenotypes of cell clones SL12.3 and SL12.4 systematized in Table 1. The expression of transcripts of the synthesis of a surface protein, carcinogenesis and tumor type was determined using Northern blot analysis, flow cytometry and in vivo injection of cloned cells syngeneic animals, respectively. 1,0 kV 1.3 kV - TCP-transcripts encode sequence (D) - J-C and V - D-J-C, respectively. Glucocorticoid response was determined by culturing cells in 1 mm dexamethasone.

Lymphoma cell was cultured in modified according to the method of Dulbecco environment, Needle, supplemented with 10% fetal of telechelic 20008 and COLO 316 cultured in RPMl-1640 medium, which has been added 5% fetal calf serum, glutamine, and 1% Fungi-bact. In the case of cells to obtain RNA cells were harvested during exponential phase growth at a density of about 5-8105cells/ml Splenocytes obtained from BALB/c mice, were sown at a density of 3106cells/ml and stimulated with 20 μg/ml Zopa two days before harvesting RNA.

Joint cultivation of cells SL12.4 and timoszyk epithelial monolayers was carried out under the following conditions. SL12.4 cells were seeded at a density so that their final concentration after three days of co-cultivation was 1106cells/ml After three days or TEL TEPI had reached a state of continuity. Cells were cultured in modified according to the method of Dulbecco environment Needle containing 10% fetal calf serum and supplemented with glutamine and penicillin/streptomycin, at 37oC.

Example 2. Study of the expression of 20.5 or PSAT-2 cell lines

In studies of the expression of 20.5 or IRU-2 used cell lines of embryonic carcinoma F9 and RSS, tumors of the pituitary ATt 20, epithelium of the thymus TEP1, epithelium of the mammary gland (ATSS 92) and F. These cells were cultured in modified according to the method of Dulbecco environment, Needle, EXT is Holocene RNA, collected during the exponential phase of growth at a density 5-8105cells/ml Splenocytes obtained from BALB/c mice, were seeded at a density of 3106cells/ml in RPM1 1640, supplemented as described above and stimulated by 10 μg/ml Sopa for 6, 24, 48, or 72 hours prior to harvesting RNA.

Example 3. Cloning and screening

As a matrix to obtain a double-stranded (DC) cDNA used poly/A/+mRNA from cells SL12.4. To pre-methylated dzanc added EcoRI-linkers. Dephosphorylation spacer elements slices gt10 ligated with cDNA and packaged in lambda phage using extract for packaging (Stratagene) according to manufacturer's instructions.

Subtraction hybridization was carried out as follows. Single-stranded cDNA was obtained from 10 mg of poly/A/+RNA SL12.4 using 250 μs 32P dCTP (Amersham) in the presence of 100 μg/ml of actinomycin D and hybridized with Rot 1260 (mol nucleotides/litres) with 25 mg poly/A/+RNA from SL12.3-cells in a volume of 8 ml, with 68oWith over 18 hours. After hybridization OC cDNA was collected by chromatography on a column of hydroxyapatite. From 1 µg of the initial SL12.4-cDNA received approximately 120 ng of cDNA (12% of the original cDNA containing 310 imp. /min) and the second of the two duplicate filter selected from SL12.4-gt 10-library was probed full cDNA, derived from mRNA SL12.3, and the second filter selection probed using subtraction-enriched cDNA SL12.4, obtained as described above. Purified by the method of plaques clones of phage were identified as SL12.4-specific through two screenings (using the separately obtained subtraction probes), after which these clones were subjected to Northern analysis, in order to ensure that they hybridize only with mRNA from cells SL12.4, but not from cells SL12.3. DNA inserts were removed from DNA by digestion with restriction enzymes HindIII and BgIII allocated for low-melting agarose (Sea Kem) and was subcloned into the plasmid vector RT/T3 (Bethes da Research Laboratory), digested with the enzymes HindIII and BamHI. These inserts could not be cut from the phage using enzyme EcoRI, since all isolates EcoRI sites were destroyed.

Example 4. Northern-blot analysis

Full cellular RNA was isolated from cell lines and tissues using guanidinoacetate method (Maniatis and others, in "Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1983)), modified as described by Wilkinson and others (EMBO J. 7:101-109(1988)). For Northern blot analysis, 10 μg of RNA was subjected to electrophoresis in 1% agarose gel containing formaldehyde, Ali by staining with acridine orazepam and hybridization with cDNA actin, SNO and/or cyclophilin. Northern blots hybridized with randomly premirovany (Amersham)32P-labeled cDNA inserts in the presence of 10% doctranslate and 50% formamide (12-18 h at 42oWith), and then Paladino washed with a final 30-minute wash in 0.1% SSPE, 0.1%of LTOs in 42oWith the 50oC. To remove the labeled probe RNA blots were washed 0,IX SSPE and 0.1% LTOs at 90oC, cooled to room temperature, drained air and kept under vacuum prior to use in subsequent hybridization. For Northern blot analysis RNA IRU-2 (20.5 or Tea) from SL12.4, SL12.3 and fresh tissue preparations obtained from Balb/-mice were isolated full cellular RNA using guanidinoacetate method. Cytoplasmic or nuclear RNA was obtained as described above for Northern analyses. 10 μg of RNA was subjected to electrophoresis in 1% agarose gel containing formaldehyde, and then transferred to nitrocellulose membranes.

Example 5. Southern blot analysis

Full cellular DNA was isolated from T cell-lymphoma, somatic cell hybrids of mouse and hamster, and tissues from other species, and then digested with restriction enzymes according to the recommendations of the suppliers.and media Nytran, then she hybridisable and washed as described above for Northern blot analysis. Southern blot analysis 20.5 conducted in accordance with the procedure described above, with some exceptions listed below. Full cellular DNA was isolated from SL12.4-liver cells of mouse and hamster, and from somatic cell hybrids. DNA from chickens and humans received in the form of commercial medicines supplied Cionetec, Palo Alto, CA. The resulting DNA was digested restricteduse enzymes, as described above. 10 μg of digested DNA was applied to each track to 0.8% agarose gel and for at least 48 hours were subjected to electrophoresis in Tris-acetate buffer, after which he blokirovala on the media Nytran, hybridized and washed as described above for Northern blot analysis. The blots containing DNA from other species were washed under conditions of lower stiffness; however, the final washing was carried out by 2 x SSPE at room temperature.

Example 6. The allocation of new cDNA clones using subtraction-enriched differential screening

Northern blots of RNA from cells SL12.3 and SL12.4 showed that the isolated cDNA clones were differentially expressed in cells SL12.3 and SL12.4, as shown in Fig.1. Insert the selected Ionia Northern blots. As shown in Fig.1, each lane contained 10 μg of full cellular RNA from cell lines SL12.4 and SL12.3. Then was probed blot containing the specified radioactively labeled cDNA insert. The arrows indicate the relative mobility of the 18S-and 28S-pPHK-transcripts. As determined by hybridization with SNO-And-probe, the number of loaded RNA SL12.3 and SL12.4 track equivalent.

Genomic DNA from cells SL12.3 and SL12.4 and the DNA of the normal liver was digested with the enzymes EcoRI, HindIII or > PST and analyzed with the use of purified inserts probes to cDNA clones. Figure 2 illustrates a southern blot-hybridization SL12.4-specific cDNA clones. Genomic DNA (10 μg per lane) from cell lines of mouse liver ACRE, SL12.3, SL12.4 and cloned T cell-lymphoma SAK, and hybrid cells SL12.3 x SL12.4, SAK x SL12.4, SAK x SL12.3, was digested with the enzyme EcoRI and analyzed by southern blot hybridization with the above cDNA clone. The side lists the sizes of the fragments in thousand base pairs (kV) defined by the joint migration HindIII-digested DNA. One or more restriction fragments were detected by probes, suggesting that the corresponding genes are present at a small number of copies were waterstove about what genes are present in the SL12.3-cell DNA in approximately the same quantities and without significant rearrangement (as shown using four different enzymes) (Fig.2). It follows that, in all probability, the differences in gene expression in SL12-cell clones due to cell-specific regulation, not by the loss of genes or their significant rearrangeable. However, this does not exclude the possibility of small rurangirwa or point mutations in SL12.3-cells. SL12.4-specific cDNA clone 20.5 (mCAT-2) was completely sequenced. The sequence of this gene is shown in Fig.3.

Example 7. Construction of cDNA library screening and sequence analysis

Construction of cDNA library and screening for the presence of a gene or 20.5 SAT-2 was carried out as described above. CDNA insert was isolated from lambda DNA by digestion with restriction enzymes HindIII and BgIII and was subcloned into the plasmid vector RT/13. Then built the restriction map and the fragments were subcloned into the plasmid RT purified on cesium chloride and directly sequenced dideoxy-method using sequenase reagents (U. S. Biochemical Corp. Cleveland. Ohio). Part of the sequence was determined with use is obtained from the UCSD Cancer Ctnter Core Molecular Biology Facility. Both DNA strands were sequenced in their entirety and the entire sequence was determined, at least in the two reactions carried out in duplicate. To collect information in overlapping sequences and to conduct an initial analysis of the DNA sequences used computer programs Microgenie. For full selection of clones received a new cDNA library using the original mCAT-2-clone. When this was received seven new clones.

Example 8. Isolation and sequencing of cDNA sequences of clone 20.5 or IRU-2

SL12.4-cDNA-library of 40,000 members in lambda gt10 was skanirovali using SL12.4-cDNA probe, subtraction against SL12.3-MPHK, and at the same time on duplicate filters using the full SL12.Can, as described above. For characterization of the gene 20.5 used screening method. Based on the characteristics of its expression cDNA clone described above was first identified Thea-genome (Te-T, ceII early activation, i.e., the early activation of T cells). Then, as described below, this gene was renamed the gene IRU-2. The insert from this clone sequenced on both strands using dideoxy-method (sequence of this gene is shown in Fig.3). Yasai stretching before 1769 p. O. cDNA does not contain a signal or polyadenylation And poly-a region. 20.5-cDNA sequence predicts the synthesis of a transmembrane protein complex structures. Figure 3 shows 453-amino acid sequence of the predicted protein molecular weight which is 49,57 kDa (unmodified). Specified DNA, obviously, contains the complete coding region, as predicted N-terminal meinenemy codon is surrounded by a consensus Kozak sequence (CXC AUGG, where X can be A, U, G or C), which is the optimal site of initiation of translation; and the predicted 5'- and 3'-noncoding region contains multiple stop codons in all three reading frames. Later, these data were checked using the new cDNA clones. The physical properties of the predicted protein was analyzed using the Microgenie program and PC. Three potential N-glycosylation sites are shown by asterisks in Fig.4-7. The predicted protein consists of 9 highly hydrophobic regions (underlined in Fig. 3), and 7 of them have properties of transmembrane domains (based on analysis using the software system software IntelliGenetics: SOAP HELIXMEM, NOVOTNY and RAOARGOS).

20.5-cDNA probe detects two transcript approximately 4.5 kV and 8.5 kV, which was present in the cells SL12.4, but was absent in cells of T-lymphoma SL13.2 (Fig.4). The analysis was performed on subcellular localization of these two transcripts in order to determine whether they are both Mature transcripts detected in the cytoplasm, or larger nuclear RNA is a precursor. It is obvious that both transcripts are fully progressirovanii RNA, because they are found in the cytoplasm (Fig.5). The origin of these two transcripts is not yet clear; they can be produced in the alternative transcription initiation and alternative splicing of the transcript or the utilization of alternative polyadenylation signals. Both Mature cytoplasmic transcript (an 8.5 and 4.5 kV) are larger than the cDNA clone. Thus, the cDNA is incomplete, although, apparently, and contain the complete coding region. Both transcript was detected using probes derived 5'-region (nucleotides 1-380) and the 3'region (nucleotides 2005-2394) cDNA clone, suggesting that this cDNA clone is not a cloning artifact, which connects the two H quantities of transcripts, present in nuclear and total RNA preparations that can be isplayeradmin or partially planned nuclear precursors (Fig.5). The results gave rise to the subsequent screening of the new SL12.4-cDNA-library and the allocation of additional cDNA clones.

Several murine cell lines derived from embryonic tissue (F9 and RSS), epithelium of the mammary gland (MIU) and nervous tissue (ATt20), were analyzed for the expression of TEM-RNA. None of these cell lines did not show significant expression of TEM-RNA. In contrast, cell lines, derived from the epithelium of the thymus (TEPI), and fibroblasts (ZTZ, F) contained mRNA Tea, albeit on a much smaller number than the cells of T-lymphoma SL12.4 (Fig.6).

In order to determine expresses whether TEM gene in normal tissues and cells of the lymphoid lineage differentiation were investigated Northern blots obtained from mRNA of mouse tissues. Cells of the thymus, inactive, spleen, lymphoid tissue associated with the gut (CALT), and bone marrow did not show significant expression of TEM-mRNA (Fig.7). However, TEM-transcripts were induced in normal spleen cells, activated T-cell mitoga proishodit cell clone specific way after proper presentation of foreign antigen. From non-lymphoid tissues only liver tissue expressed a moderate amount of Tea-mRNA. Thea-transcripts were not detected in the intestine, stomach, ovaries (Fig.7), brain, heart, lung, kidney, pancreas, or testicles. Thus, expression of TEM gene is limited to only certain types of cells, such as cells activated spleen, epithelial cells of the thymus, the cells T-cell lymphoma and liver cells.

Example 9. The induction of Tea-mRNA

RNA was obtained after 6, 24 and 72 hours after addition of Con a to the splenocytes. Figure 8 shows the kinetics of induction TEM gene in activated splenocytes. Performed Northern analysis of RNA from unactivated and activated spleen cells collected through the above periods of time after activation of the T-cell mitogen Con A. In Fig.8 final interval after addition of Con A is 72 hours, and in Fig.9 shows the different blots RNA together with the control sensing cyclophilin (WM) to illustrate the relative amount of mRNA loaded in each lane. Unlike WM-mRNA loading rRNA in each lane was identical, as was shown by staining with acridine orange. Thea-transcripts were not detecting the PoE reached the maximum value after approximately 48 hours. Although the full amount of RNA in each lane the same way (10 µg) (as shown by staining with acridine orange), but the ratio of ribosomal RNA to mRNA, obviously, changes in the activation of T cells, since the amount of actin, SNO-And - ticlopidinesee of transcriptof (10 µg total RNA) increases (Fig.8). While inducing the expression of TEM gene in comparison to the control RNA in the activation of T cells is evident.

Example 10. Homology 20.5-DNA and AA sequences with mouse ERR

The search for homologous sequences carried out using a database of Bionet not found any significant similarities between 20.5-cDNA and other previously known DNA sequences. However, comparison of this sequence with the cDNA clone of mouse ecotropica retroviral receptor (ERR) showed significant sequence identity. The gene that encodes ecotropic retroviral receptor, was designated Rec-1, a now renamed IRU-1. Figure 10 field ERR - 20.5-cDNA sequences included in the comparative analysis are marked by vertical lines. For each cDNA are indicated open reading frames. Figure 11 full cDNA-posledovatelyam in ERR-cDNA sequences (p. O. 400-2425) no, the first 400 p. O. horizontal lines indicate positions in which the sequences are identical. This comparison was performed using the software Microgenie and with appropriate gaps that have been made to identify sequences with the highest similarity. The percentage identity was calculated only for clearly overlapping cDNA regions. 20.5-cDNA is much longer at the 3'-end and ERR-cDNA is significantly longer at the 5'-end.

In the diagram, depicting two cDNA shows that ERR-longer cDNA 5'-end, and 20.5-cDNA significantly longer at the 3'-end; and these two cDNA are similar along its entire length. The coding region is indicated by the shaded part of each cDNA clone. Comparative DNA analysis of primary structures showed that the identity of the DNA sequences between the overlapping areas 20.5-cDNA (p. O. 1-2047) and ERR-DNA (p. O. 400-2425) is 59%; and one area 20.5-cDNA (p. O. 1011-1088) is identical to 80%. 5'-noncoding region 20.5-cDNA sequences (p. O. 1-410) is 68% identical overlapping 5'-coding region ERR-cDNA sequences, suggesting that these two genes originated from a common placenta is ERR-protein, derived from the ERR-cDNA sequences. A comparative analysis of the TEM-protein ERR-protein found two areas with a high degree of similarity of amino acids (Fig. 7). Figure 7 illustrates comparative sequence analysis of the TEM-protein sequence of murine ecotropica retroviral receptor. Figure 7 schematically shows the comparison of the two predicted protein products; Tea-protein extends from 1 to 404 amino acids, and ERR-protein ranges from 204 to 603 amino acids. Figure 13 amino acid sequence of the predicted protein encoded by 20.5-cDNA, shown in the top row, and the proteins encoded by ERR-cDNA - on the bottom line. Two areas are marked in bold parentheses in Fig.13, reveal a high degree of identity: Region 1 (192 amino acids) showed identity 81,3%, and Region 2 (79 amino acids) showed identity with 51.9%. Area 1, indicated in parentheses, discovers the identity sequence is 81% and the similarity of amino acids (for 193 and.K.) 91%. Region 2 has a sequence identity of 62%, and the similarity (over 60 amino acids) by 75%. Differences conservative amino acids specified by two points between them, and the identity of aminoisoquinoline and characterization solutionsa cell hybrids "of the Chinese hamster X mouse" described Hogga and others, J. Virol., 62: 1055-1056 (1988). In short, the mouse strain NFS/N was obtained from the Division of Natural Resources, NIH, Bethesda, MD. House mice (Mus Musculus) were obtained from laboratory colonies originating from mice originally caught in Skive (Denmark) and kept under the supervision of Dr. M. Potter (NCl, NIH, Cjntract NOI-CB2-55894) in the laboratory Hazelton Laboratories, Roclville, MD. Female hybrid mice NFS/Nx x m." were subjected to reverse mating with male house mice, resulting in the experimental animals. From the liver of these mice were extracted DNA, which is then digested by enzymes Sacl and Bam HI were subjected to electrophoresis in a 0.4% agarose gel for 48 h at 24 and transferred to nylon membranes (Hybond N+, Amersham). Membranes were hybridisable with [32P]-labeled 20.5-cDNA and 438 p. O.-probe representing a gene DNA polymerase, present on chromosome 8. After that, membranes were washed and probed. The samples taken from kidneys of the same mice were identified by inheritance markers Cu-I and Es-I by histochemical staining after electrophoresis in starch gels.

Because ERR-gene product functions as a viral receptor, we carried out mapping of TEM gene in order to determine if it is present on brskalnik different somatic cell hybrids, formed between the cells of the Chinese hamster and mouse, kept a limited number of different mouse chromosomes. These hybrids were used for mapping TEM gene. Southern analysis of DNA obtained from hybrid cells and digested by the enzyme Pst I, showed that 7 out of 21 hybrids contained the mouse-specific DNA fragments. Comparison of the known content of mouse chromosomes by positive hybridization with the mouse-specific DNA fragments showed the best correlation with mouse chromosome 8 (table 2).

Mapping Tea-gene was performed using somatic cell hybrids "mouse-hamster". The symbol (+/) indicates the presence, and the symbol (-/) no restriction Tea-fragment relative to the presence (+/) or absence (-/) specific mouse chromosomes (indicated by the number in the left column), detected by hybridization with a 20.5-cDNA probe. The number of contradictory observations is the sum of (+/-) and (-/+)-observations.

None of these hybrids was not karyotypical. They were typed for other markers, so maybe (+/-)-hybrid cell contains fragments of chromosome 8, or a small percentage of cells contains this chromosome. (-/+)-Cell can is by analysis by interspecific back-crossing was installed, what Thea gene is located on chromosome 8. Sst-1 digested DNA of mice NFS/N was produced cross-reactive bands 10,0, of 7.4 and 5.5 KB. DNA from male house mice were produced fragments 10,0; at 6.4 and 5.5 KB. Figure 9B shows a picture of hybridization 20.5-cDNA-probe experiment with reverse breeding "NFS/N x M. m.mus. FI - mouse male house mice (M. m. mus. ). Picture segregation specified polymorphism of restriction fragments with other markers on chromosome 8 suggests that this gene is associated with SG-1 and Es-1 in the following order: centromere-Polb-Cr-I-Tea-Es-I (Tables 3 and 4).

Localization TEM gene on chromosome 8 suggests that this gene is different from the gene Rec-1, which encodes ERR, and is located on chromosome 5. The data also exclude the possibility that Tea is a gene that encodes a retroviral factor receptor MCF and located on chromosome 1.

Example 12. Obtaining cDNA for microinjection

Thea-cDNA was directly subcloned into the plasmid pSP72 (Promega) to the 5'ends were connected with S6-promoter. Thea-pS P72 plasmid was linearizable 3'-end of the coding region using enzyme Styl and transcribable in vitro using S6 polymerase (Promegf, Madison, WI) in the presence of 0.3 scripty were analyzed on agarose gel and the results of this analysis were obtained dimensions crnc: a 2.5 KB (Tea) and 1.9 and 2.4 KB (mCAT-1). crnc were extracted with a mixture of phenol and chloroform (1:1) and chloroform, precipitated with ammonium acetate and isopropanol, dissolved in DEPC-treated water (1.0-4.0 mg/ml) and used for injection into Xenopus oocytes.

Example 13. Microinjection in Xenopus oocytes

In African clawed frogs (Xenopus leavis) surgically removed oocytes, which are then within 2 hours were treated with collagenase (1.28 mg/ml) type 1A (Sigma, St. Louis, MO.) in not containing calcium solution Barta (see below), washed not containing calcium solution Bart and incubated for 2 hours at not containing calcium solution Barth. Of these oocytes had to manually delete the follicles and incubated overnight at 18oWith the solution Bart: NaCl (88 mm), KCl (1 mm), Panso3(2.4 mm), HEPES (15 mm), Ca(NO2)24H2O (0.33 mm), CaCl22H2O (0,41 mm), MgSO47H2O (0,82 mm), gentamicin (10 μg/ml), pyruvic acid (3.1 mm), then in these oocytes were injected with 30-200 ng crnc 50 nl of water. Then the oocytes were incubated at 18oWith the solution of Bart in 2-3 days.

Example 14. Electrophysiology

Transport currents in oocytes was detected at approximately 60 mV using 2-microelectrode method of fixing voltage. the slots. The amino acid was dissolved in the solution to register at pH 7.3 and was introduced by changing the bath, using the recording chamber with a constant flow. Individual oocytes were obtained in several

various concentrations and were administered for periods of time for 30 seconds before and after washing registering saline, containing no amino acids. Currents were measured by the method of fixing the voltage using the Axoclamp-2A (Axon Instruments, Foster City, CA), a signal was tracked through thermoresistibile device. Used oocytes had initial residual voltage of at least about 46 MB. To measure in the absence of sodium, in the nonvolatile salt solution, NaCl was replaced with equimolar amounts of holdingarea. Control oocytes were obtained from the same batch of cells, and that the subjects oocytes. In these control oocytes were injected with either 50 ml of DEPC-water or 50 ml in vitr-transcriptional G Iu P3-crnc.

Example 15. Surgical operations on animals

Partial hepatectomy was carried out by longitudinal represa so that anastasiasunny (70 mg/kg phenobarbital-sodium) mouse ACRE (aged 6-8 weeks) were removed approximately 60% of the liver, Then izvlecheniyu of liver tissue after resection, used as a temporary zero control. Nine hepatectomized animals mice were analyzed (three mice for each time interval). In this analysis included six falsely operated animals.

Example 16. Northern analysis

Full tissue RNA was obtained from mice ACRE, and RNA from the tissue of the testes were obtained from the "Nude" mice Balb/c mice. To do this, the tissue was rapidly collected, homogenized in a 10-fold excess (wt./volume) buffer for lysis of tissue, and total RNA was obtained using the one-step method of extraction with phenol. RNA was stored in an aqueous solution containing 5% 2-mercaptoethanol, 5 mm 3D TA and 0.5% Sarkosyl, at -70oC. Cell lines were cultured in modified according to the method of Dulbecco environment Needle (DMEM with high glucose content), to which was added 1% glutamine (at 37oC) in 5% CO2. Cells were washed in phosphate-buffer solution was literally 10-TEW volumes of buffer for Alisia tissues and received total RNA.

Total RNA (10 µg) from each tissue analyzed (in duplicate) on a simple denaturing formaldehyde/agarose gel and transferred to a membrane carrier NitroPlus (MSI, Westboro, MA, 01581). Prints probed randomly praimirovanie sweep BamHI/Pvu II digestion of source 20.5-cDNA-clone. One half of the duplicate Northern blot was probed with a mixture of IRU-2 (2,1 kb-pJet-fragment) and cyclophilin (0,7 KB), and the other half simultaneously probed Thea-cDNA of (2.4 KB) and cyclophilin using identical conditions for hybridization and probes with equivalent potency. Then the blots were washed together in the final stiffness (0,1 SSPE, 0.1% of LTOs, when 42oWith over 30 min) and exposed to film for the same period of time.

Example 17. The transport of cationic amino acids in micro-injected the oocyte

Figure 10 depicts a histogram showing the response cDNA - and water-injected oocytes Xenopus 20 main amino acids required for protein synthesis, as well as ornithine, homoserine, and MeAlB [alpha-(methylamino)somalina acid; specific substrate system]. The transport was assessed by determining changes in membrane current induced by amino acids. Oocytes injected with TEM-crnc generated significant internal currents in the presence of arginine, lysine and ornithine. Thus, TEM-crnc encodes a protein that mediates specific transport these cationic amino acids. On this basis, the gene Tea renamed gene mCAT-annih controls, suggest permanent presence of small endogenous transport activity, since the internal currents of less than 1 are observed for most amino acids, and the maximum value of this current does not exceed 5 (Fig.16). Moderate current was also observed upon stimulation of oocytes histidine (at pH of 7.3). It is possible that the observed transport a small amount of histidine (Fig.16) due to the insignificant number of histidine, which is cationic at pH of 7.3. It is possible that these cationic carrier is recognized only the protonated form of histidine. In addition, the transport of lysine and ornithine, mediated IRU-1 and mCAT-2, was virtually indistinguishable.

For the evaluation of stereo-specificity of transport was the comparison of L - and D-forms of arginine, lysine and ornithine. From Fig.17 shows that L-arginine, L-lysine and L-ornithine concentration of 1 mm generate internal currents, which are 8 times higher than the currents generated by the D-isomer. Thus, mCAT-2-mediated transport is specific in relation to biologically active forms of the amino acid substrates.

Example 18. mCAT-2-mediated transport is the sodium-independent

Carriers of amino acids, of oocytes was evaluated need for the presence of sodium ions for transport of arginine, lysine, ornithine and homoserine. For this purpose, in order to maintain osmolarity in solution for registration, nutrigold was replaced by choline chloride. As can be seen from Fig.18, the transport of arginine, lysine and ornithine occurs regardless of the presence of extracellular Na+. In contrast, the transport of dipolar homoserine is a considerable degree of sodium-dependent (p=0,0025), although the values of the transport currents are very small (+system mediates the transport of cationic amino acids, which does not depend on the presence of sodium; however, this presence is necessary in the case of import dipolar amino acids, such as homoserine).

Example 19. The affinity for the substrate and the saturation IRU-mediated transport

Carriers of amino acids detect the characteristic properties of substrate saturation. Saturation is observed at a concentration of 1 mm arginine, lysine and ornithine and 10 mm histidine (Fig.19). As expected for a system+vectors of the amino acid concentration required for the transport of such cationic amino acids like arginine, lysine and ornithine were lower than the concentration required for the transport of histidine.

Seeming TOmvalues for arginine, ornithine presented in Table 1. The specified value TO themwere determined using nonlinear regression analysis of the dose-dependent response (see box in Fig.19) using GraphPad-programme InPlot 4.03. mCAT-2 protein was detected apparent affinity for arginine, lysine and ornithine in a higher degree than histidine (Fig.13). Standard flexible on average did not exceed 2.0 for arginine, 2,4 for lysine and ornithine and 3.8 for histidine. Apparent values of Kmare given in Table 5.

Seeming TOm-values for the IRU-2 were obtained on the basis of the data presented in Fig.19, andm-values for the IRU-1 were obtained in an identical way using the GraphPad program InPlot 4.03. The value Kmfor IRU-1 was determined using 13 oocytes; and the value Kmfor IRU-2 was determined using 17 oocytes for arginine, 7 oocytes for lysine, 8 oocytes for ornithine and 3 oocytes for histidine. The oocytes were injected 30-185 ng cDNA.

Seeming TOmthe values obtained for each amino acid were approximately the same for different oocytes, whereas the value of Imaxtended to fluctuations. The variation of the values of Imaxprobably due to different levels of expressiom, values of I for different amino acids, derived from a single cell, consistent with each other. Maximum currents generated in response to this amino acid not find noticeable differences, and one of the oocyte, and the deviation of the maximum current value from the average value is less than 3.5 on for arginine, lysine, ornithine and histidine.

TOmvalue (0,187) for mCAT-2-mediated transport of arginine significantly higher than the initial value for LH CAT-1 (0,07-0,077). Therefore, in order to verify the difference of the affinity of the two proteins in relation to arginine was determined the apparent affinity of the IRU-1 protein relative to arginine in the oocytes. Table 5 presents the values FORmobtained from experiments on the comparison of arginine transport mediated or IRU-1 (0,206 mm 0,02 cf. square Osh.), or IRU-2 (0,187 0,028 mm cf. square Osh.).

Example 20. Expression of the IRU-1 - mCAT-2-PHK in tissues and cell line

Figure 20 shows the expression of the IRU-1 and IRU-2 genes, assessed using Northern blot analysis. mCAT-1 mRNA was detected in all 15 tissues except liver. No IRU-1 protein in the liver caused by the inability to ecotropic retroviruses to infect this kind of fabric is in trace quantities, in the skin and skeletal muscle (only after prolonged exposure).

As for the mCAT-2-PHK, the most abundant expression was observed in the liver, and high levels of expression were observed in skeletal muscle. Detectable expression of mCAT-2 transcripts was observed in the tissues of the stomach, skin, brain, lung, and uterus, and in the tissues of the testicles, ovaries and heart were observed only trace amounts of mCAT-2-mRNA. IRU-2 mRNA was not detected in resting lymphocytes of the spleen, the colon and the small intestine, thymus, and kidney.

All of the 15 analyzed tissues expressed either one or two IRU gene. IRU-1 - mCAT-2-TRANS crypt simultaneously expressibility in several types of tissues, namely, in the tissues of the uterus, brain, and lungs. Co-expression of these genes was observed in cell lines of T-lymphoma SLI2.4 and two cell lines hepatoma (Fig.20). In the Mature tissues of the body, one gene is usually dominant. For example, in the liver is expressed only mCAT-2 mRNA, whereas mCAT-1 mRNA is detected only in the tissues of the intestines, in resting T-cells, spleen, and thymus.

Example 21. In normal and regenerating liver observed expression GSAT-2-, but not m SAT-1 mRNA

ery is mitogen-active state. Was investigated RNA derived from liver of control, lonaprisan, and partly hepatectomized animals, and assessed the expression of both IRU genes (Fig.20). mCAT-1-mRNA was absent in the liver, but was rapidly induced under cultivation of liver cells; and was present in two cell lines hepatoma (see Fig.21). mCAT-1-PHK was not detected in the control and in regenerating liver and in the liver lonaprisan animals. In contrast, expression of mCAT-2-PHK was observed in the control and regenerating liver, and liver lonaprisan animals, and this expression was not induced when a significant change of conditions under the influence of massive mitosis, which were subjected to liver cells 24 hours after hepatectomy (Fig.21B).

The original isoform IRU-2 expressives in normal tissue, and specific probe for this region was obtained from the cDNA (Fig.S). Northern analysis (Fig. 210 showed that this isoform IRU-2 really is expressed in all six of the tested tissues.

Example 22. Characterization of mCAT-2-protein

Each mCAT-isoforms contain specific sequences that give each protein-transfers IRU-2 have the same COOH-terminal sequence, which is completely different from terminal sequence IRU-1 and longer than her 31 amino-acid. mCAT-2 protein contains a carboxy-terminal (55 amino acids) hydrophilic region next to the last transmembrane domain and the contiguous as expected with the cytoplasm (MSI-3). This is enough to make interaction with other intracellular proteins for signal transmission or generation of channels for the passage of the transported amino acids in this intracellular pool. If the specified carboxy-end is intracellular, it can be associated with arginase in each tissue, but predominantly in the liver. The liver can Express the protein carrier (IRU-2A) with a very high level TOmin periportal cells, which helps prevent the depletion of arginine (or lysine) from portal blood. Regardless of whether the carboxy-end of the extracellular or intracellular, he may participate in the binding of other accessory molecules known that they also participate in the transport of amino acids.

The second distinctive feature is the AA-binding domain mCAT-2 protein. Two isoforms: IRU-2 are identical to the is of identity over the entire sequence 97%. However, the option IRU-2A has TOmwhich is 10 times TOm(when measured in Xenopus oocytes). In contrast, the homology sequences IRU-1 and IRU-2 is only 61%, despite the fact that these two vectors are identical TO themand by expression in Xenopus oocytes, they are functionally indistinguishable. So, basically, low affinity IRU-2A with respect to arginine may mean that he performs other physiological functions.

Example 23. The production of antibodies

Antibodies were produced according to the methods Billetta and Zanetti Immunomethods 1: 41-51 E). Basically, this method consists in the fact that the double-stranded oligonucleotide encoding specific epitope is administered in a highly variable region (CDP3) chain of the Mature immunoglobulin, resulting in this area becomes "anticensorware". In this case, were selected oligonucleotides, representing divergent sequence IRU-2 and IRU-2A. These sequences and their length was chosen according to three criteria: 1) antigenic potential; 2) sufficient difference from each other to have produced antibodies specific for each epitope;

3) they must be entered in the variable region chain of IgG.

For comparison, no IRU (Fig.22):




**. **. **... *** *****. **....... ** **... *...

"x" indicates identical residues; "." indicates a conservative substitution. Region of the protein corresponding to the fragment of alternative splicing, is and. K. 360-400. The scope for anti-generowania or antibody-based test designs is the amino acid 377-388. In addition, in Fig.22 shows the sites used for the synthesis of gst-mCAT-2-hybrid proteins, namely:

(1) IG or amino acids 1-33;

(2) 2V or amino acids 141-166;

(3) 3C or amino acids 212-243;

(4) 4L or amino acids 438-490; and

(5) 50 or amino acids 604-674.

A synthetic oligonucleotide encoding the sequence IRU-2, shown above in bold lines, hybridized with the complementary synthetic oligonucleotide, and was introduced in SOC-region accordingly rearanging mouse YN62 gene. Restriction sites were designed so that the oligonucleotide was integrated with maintaining their own frame of scitable or reading frame of the heavy chain. Orientation and reading frame was confirmed by sequencing. Full SOC-region, th gamma-1 and contained in expressroom the vector pN-gamma I mammal. The final design contained a chimeric sequence IRU/DNA heavy chain of mouse IgG/human; a gene of resistance to neomycin for selection of cells transformed mammals, and the gene for resistance to ampicillin for selection in bacteria. Were also constructed in a similar design of oligonucleotides encoding the IRU-2A - IRU-1-specific sequence. In addition, there was obtained the fourth sequence for IRU-2, localized in the 3'-end of the coding sequence.

Each construct was introduced by electroporation into mouse cells J 558L, which constitutively Express a light chain lambda-1 mouse. G418-resistant cell clones secreting engineered antibody, selected and multiplied. Secreted anticensorware antibodies were purified and used for production of antisera in accordance with the traditional scheme of immunization.

Rabbit anticigarette was purified using sequential affinity chromatography. This anticigarette inflicted on sepharose associated with gamma-1 man removal of antibodies against the constant region of the gamma chain-1 person, then on sepharose associated with murine VH62 removal and the bodies which specifically recognize a separate SAT-epitopes. The specificity of the antibodies are then examined by Western analysis GST/mCAT - and native IRU-proteins. Lysates SL2.3 and SL12.4 was used as a source of native proteins. Cells SLI2.3 were used as controls because they Express the IRU-1, and does not Express an isoform of the IRU-2.

Example 24.

Anti-MSAT antisera can be injected into a patient or animal after mixing with the pharmaceutical carrier. Pharmaceutical carriers that contribute to the delivery of antibodies to the affected tissues of an individual well known to specialists in this field. When used in therapy in vivo anti-MSAT antisera truly will be applied in therapeutically effective amounts, i.e. in amounts which can effectively inhibit the transport of cationic amino acids in the body undergoing treatment of an individual or animal. One way in which the antisera can be entered, is intravenous. The amount of injected antisera will usually be in the range of approximately 0.1 to approximately 100 mg/kg of body weight of the patient. Intravenous immunotoxins mogote pharmaceutical carriers for intravenous represent water, saline, ringer's solution, dextrose 5% solution of human serum albumin. Can also be used non-aqueous media, such as non-volatile oil and etiloleat, as well as liposomes. The medium may contain minor amounts of additives such as substances that enhance isotonicity and chemical resistance, for example, buffers and preservatives. Other pharmaceutically acceptable carriers can be used for other routes of administration, and is well known to specialists in this field.

1. Rabbit anticavity, inhibiting the transport of cationic amino acids, induced against T-cell protein encoded by the genome of the IRU-2 and having the amino acid sequence of SEQ ID 2.

2. The pharmaceutical composition inhibiting the transport of cationic amino acids and containing an immunologically effective amount of antisera under item 1 in a pharmaceutically acceptable carrier.


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