Urocortin proteins and their using
FIELD: genetic engineering, medicine.
SUBSTANCE: invention relates to isolated DNA encoding human peptide related to urocortin and peptide named as urocortin II. These peptides are relative with corticotropin-releasing factor and involves in mechanisms for initiation of hypophysis-suprarenal responses for the stress. Pharmaceutical composition comprising such peptide in combination with acceptable vehicle can be used in treatment of pathophysiological states, such as enhanced body temperature, appetite disorder, congestive cardiac insufficiency, stress, anxiety state and undesirable low levels of ACTH.
EFFECT: valuable medicinal properties of urocortin proteins.
33 cl, 27 dwg, 2 tbl, 16 ex
The premise of the INVENTIONS
DESCRIPTION FEDERAL FUNDING
This invention was partially made using funds received from the Federal government under grant No. DK-26741. Thus, the Federal government has certain rights to this invention.
The SCOPE of the INVENTION
This invention in General relates to the fields of neuroendocrinology and mechanisms involved in the stress. More specifically, this invention relates to new peptides, related corticotropin-releasing factor, urocortin II and related urocortin protein of a person involved in the response to stress.
DESCRIPTION RELATED FIELD
The corticotropin-releasing factor (CRF) is a peptide consisting of 41 amino acids, it is best known because of its important role in the initiation of the pituitary-adrenal responses to stress, effect, mediated by CRF receptors type 1 (1). In addition, corticotropin-releasing factor is widely distributed in the brain, and repeatedly has been shown that it is involved in the mobilization of complementary Autonomous and adaptive behavioral reactions in the case of many threatening conditions (2, 3). This prompted pull the widely accepted hypothesis that the corticotropin-releasing factor family of related PE is the Chida play an important role in the regulation of the hypothalamic-pituitary-adrenal axis (hPa) in the major and stressful conditions (4, 5). It is also believed that the corticotropin-releasing factor is also involved in other neuroendocrine and paracrine responses in many tissues. Members of the CRF family are integrating endocrine, autonomic and behavioral responses to stress factors. These peptides may also be involved in the control of appetite, arousal, and cognitive functions. In the long-term impact of stress you may experience severe psychological and physiological effects such as anxiety disorders, anorexia nervosa and melancholic depression.
Family members of the corticotropin-releasing factor mediates its biological actions by specific binding to CRF receptors with high affinity (6, 7). The CRF receptors are receptors linked to G-proteins that act through adenylate cyclase and are structurally related to the secretin family. The specified collection also includes GRF, VIP, PTH, and calcitonin receptor. Gene CRF receptor has 13 exons, and found several splicing variants of this receptor. The receptor for CRF-R1 distributed throughout the brain and is found in places of transmission of sensory and motor signals (8). CRF-R2α distributed in the lateral septum, ventral-medial hypothalamus, nucleus of the solitary path and dorsal is the core of the seam, they represent areas where CRF-R1 is expressed very little or not expressed (9). CRF-R2β mainly found in the peripheral areas, including heart, blood vessels, gastrointestinal tract, epididymis, lung and skin (7, 10). Pharmacology of two types of receptors differs in that the corticotropin-releasing factor has a low affinity to CRF-R2 (Ki=15-100 nm), but a high affinity to CRF-R1 (Ki=1-2 nm). Other related peptides, such as urotensin carp, sauvain frogs and urocortin have high affinity for CRF-R2. Mouse, knocked out by CRF-R2, manifest increased like disturbing behavior, caused by hypersensitivity to stress factors (11).
It was found that in some groups of cells, identified as the scene of peptides that cause stress such autonomic and behavioral responses, absent or weak expression of the required ligand(Dov), receptor(ROS) or both (12, 13). This has led to the intensification of the search for more related CRF signaling molecules, which currently consist of two ligand associated with G-protein receptors, derived from two separate genes (CRF-R1 and CRF-R2), and binding protein whose function is unknown until the end (14, 15).
Recently discovered second related neuropeptide CRF mlekovita them, urocortin (Ucn), and showed that he with high affinity associated with both known types of CRF receptors, whereas CRF with a greater preference associated with CRF-R1. When the Central introduction urocortin more effective than CRF, appetite suppression, but less effective in generating such acute anxiety effects and generalized activation behavior (17). It is used to show that urocortin may at least partially mediate some stress-related effects that were first attributed to CRF, serving as an endogenous ligand CRF-R2. However, this view has been questioned as a result of such observation, as the observation that the main cellular location of expression of urocortin in the brain are not recognized as integral components of the Central stress related schemes and that most of the major sites of expression of CRF-R2 poorly innervated containing urocortin processes (18). These and other data support the possible existence of one or more ligands of CRF receptors in the mammalian brain.
The prior art does not meet the requirements due to the lack of identified additional genes and proteins urocortin. This invention fulfills this long-standing need and needs is in this field.
Rapid progress in the accumulation of data on the sequences of the genomes of human and mouse allows for the identification of new representatives of different families of proteins. A new peptide sequence related urocortin peptide person (URP), identified in the published database of the human genome. The sequence of a peptide related urocortin, has homology with urocortin man (44%), urotensin carp (39%) and CRF man (36%). Synthesized peptide, related to urocortin, with a higher affinity binds to CRF-R2 (Ki=0.5 nm)than with CRF-R1 (Ki=70 nm). Peptide person related urocortin, stimulates the secretion of ACTH from the cells of the anterior pituitary gland of rats, although much less efficiently than urocortin or CRF. Using search homology sequences also identified a mouse gene that encodes a peptide of 38 amino acids, which represents a new member of the CRF family of neuropeptides. The specified peptide called urocortin II (Ucn II)differs from other known representatives of the family of the fact that it binds with high selectivity with CRF-R2. Evidence of urocortin II in the rat brain obtained by immunohistochemical studies and research in situ hybridization using antihelicobacter towards urocortin II.
In one embodiment, the present invention presents the DNA sequence encoding urocortin II. The sequence can be selected from the group consisting of: an isolated and purified DNA which encodes urocortin II; an isolated and purified DNA, which in conditions of high stringency hybridized with antisense sequences, complementary DNA urocortin II (conditions of high stringency is defined as the flushing of the membrane at high temperature and low salt concentration, is functionally equivalent to 0.1 x SSC at 65° (C); and an isolated and purified DNA encoding urocortin II, but which differs in sequence due to the degeneracy of the genetic code. Specified DNA preferably encodes the precursor protein having the amino acid sequence shown in SEQ ID NO: 10.
In another embodiment of the present invention the direction of the present invention is a vector that can Express urocortin II. Such a vector comprises DNA that encodes urocortin II, and the regulatory elements necessary for expression of urocortin II in the cell. In a preferred embodiment, the specified vector encodes a protein with amino acid sequence SEQ ID NO: 11. The direction of the present invention is a host cell, transfusiona this vector is expressing urocortin II with the specified vector. Protein can be expressed in a cell type selected from bacterial cells, mammalian cells, plant cells and insect cells. In one preferred embodiment, the protein is expressed in E. coli.
In yet another embodiment, the present invention relates to isolated and purified protein of urocortin II human encoded by the above DNA. Preferably cleaned related urocortin peptide person has an amino acid sequence corresponding to SEQ ID NO: 11.
In another embodiment, this invention relates to an antibody directed against the protein of urocortin II. The specified antibody can be a monoclonal antibody.
In another embodiment, this invention relates to a pharmaceutical composition containing the protein of urocortin II. Such pharmaceutical composition can be used to reduce the temperature of the body, suppress appetite, and treatment or prevention of congestive heart failure and various disorders associated with stress.
In the following embodiment, the present invention relates to DNA sequences coding related urocortin peptide person. The sequence can be selected from the group consisting of: an isolated and purified DNA which encodes a related urocortin peptide man; stand-alone and the untreated DNA, which in conditions of high stringency hybridized with antisense sequences, complementary DNA related urocortin peptide person (conditions of high stringency is defined as the flushing of the membrane at high temperature and low salt concentration, is functionally equivalent to 0.1 x SSC at 65° (C); and an isolated and purified DNA encoding related urocortin peptide person, but which differs in sequence due to the degeneracy of the genetic code. Specified DNA preferably has the sequence shown in SEQ ID NO: 1 and encodes the precursor protein having the amino acid sequence shown in SEQ ID NO: 2.
In another embodiment of the present invention the direction of the present invention is a vector that can Express the related urocortin peptide person. Such a vector comprises DNA that encodes a related urocortin peptide person, and the regulatory elements necessary for expression related urocortin peptide man in the cage. In a preferred embodiment, the specified vector encodes a protein with amino acid sequence SEQ ID NO: 3. The direction of the present invention is a host cell, transfusiona this vector and expressing the related urocortin peptide person with specified what the sector. Protein can be expressed in a cell type selected from bacterial cells, mammalian cells, plant cells and insect cells. In one preferred embodiment, the protein is expressed in E. coli.
In yet another embodiment, the present invention relates to isolated and purified protein related urocortin peptide person, encoded by the above DNA. Preferably cleaned related urocortin peptide person has an amino acid sequence corresponding to SEQ ID NO: 3.
In another embodiment, this invention relates to an antibody directed against a protein related urocortin peptide person. The specified antibody can be a monoclonal antibody.
In yet another embodiment, the present invention relates to pharmaceutical compositions containing protein related urocortin peptide person. Such pharmaceutical composition can be used to reduce the temperature of the body, suppress appetite, and treatment or prevention of congestive heart failure and various disorders associated with stress.
In another embodiment of the present invention described various modifications of proteins urocortin II and related urocortin peptide of human rights, including the modification of the sequence and individual amino acids of proteins. Modification targetlocale the pairing urocortin II and related urocortin peptide person with fluorescent labels, complex radionuclides and toxins.
BRIEF DESCRIPTION of DRAWINGS
In order to achieve a detailed understanding of the above features, advantages and objects of the invention, as well as other issues that will become apparent, it is possible to imagine a more particular description of the invention, the essence of which is briefly described above, with reference to some of its variants, which is illustrated by the accompanying drawings. These drawings form part of the description. However, it should be noted that the accompanying drawings illustrate the preferred variants of the invention and therefore should not be considered limiting in data frames.
The figure 1 shows the sequence of human genomic DNA, on the basis of which predicted the existence of a new peptide, related to urocortin and CRF. Genomic sequences identified in the published database and used to calculate a new sequence related urocortin peptide person. The alleged site of the start position is 1, and the sequence of the Mature peptide shown in bold. Designed the sites of cleavage of the signal peptide is indicated by arrows.
The figure 2 shows the presumed progenitor related urocortin peptide person. The underlined region represents Nepal the th cDNA sequence, which was isolated by PCR from a cDNA library of the pancreatic islets of the person.
The figure 3 shows the comparison of related urocortin peptide person (URP) (SEQ ID No4) with human Ucn (SEQ ID No:5), urotensin I (SEQ ID No:6), CRF (SEQ ID No:7), sauvagine frog (SEQ ID No:8) and CRF/Uro sea dogs (SEQ ID No:9). The area of highest homology are within the white rectangles. Specify the number of conservative amino acids.
Figure 4A shows the deduced amino acid sequence of Ucn II. The starting methionine, bold, localized above the coding region of the peptide, which is limited by the frame. The complete nucleotide sequence was placed in Genbank (inventory No.AF331517).
Figure 4B shows the alignment of Ucn II mouse (SEQ ID No:10) with homologous peptides of human (SEQ ID No:11) and fish (URP)(SEQ ID No:12) and Ucn rat (SEQ ID No:13) and CRF rat/human (SEQ ID No:14). Residues identical to the sequence Ucn II mouse is limited to the frame. Ξ indicates amidation site.
In figures 5A and 5B shows indirectly related urocortin peptide person substitution125I sauvagine, communicating with CRFR1 and CRFR2β. The affinity of Ucn and peptides URP in relation to CRFR1 and CRFR2β, stably expressed in cells SNO, was determined by competitive replacement125I sauvagine. Presents data from experiment 3 is in, and the values of the dissociation constants/inhibition (Ki) (95% confidence interval) was calculated using the program Prism.
In figure 6 (a-C) shows the expression of mRNA of urocortin II in the rat brain. The dark-field micrograph showing the marking (white granules)observed in some areas when using labeled with isotopes probe antisense crnc derived from cDNA of urocortin II mouse. Positive hybridization signals are seen in paraventrikulyarnoe nucleus of the hypothalamus (figure 6, A), mainly in its magnocellular division (pm), while a more diffuse signal is visible in parvocellular region (mp), and extensively in the blue place (locus coeruleus) (LC; figure 6), the motor nucleus of the facial nerve (VII, figure 6) and cerebral meninges (men) on the ventral surface of the brain. Other abbreviations: CBL, cerebellum; v3, third ventricle; v4, fourth ventricle. Increase: figure 6, a and b, H; figure 6, C, X50.
The figure 7 shows autoradiogram expression related urocortin peptide of human rights in the Primate hypothalamus. PVH, paraventrikulyarnoe kernel; SO, supraopticus core; CN, caudate nucleus; och, optical chiasma; me, median Eminence; ac, anterior commissure; ic, internal capsule; Sept, partition.
In figures 8 (a-F) showing the pattern of cell activation in response to Central microinject is the Oia urocortin II. Figure 8, a-C and E: the bright field Micrograph immune-peroxidase preparations, showing induced expression of Fos in rats scored 2 hours after intracerebroventricular (icv) injection of 1 μg of synthetic urocortin II mouse. The dark-field micrograph showing the histochemical data on the localization of hybridization of mRNA CRF-R2 in the areas corresponding to the areas shown on figure 8, C and E, are presented in the figure 8, D and F, respectively. Central injection urocortin II causes the induction of Fos, mainly in the group of interconnected structures that are involved in Central autonomic and neuroendocrine regulation, including parvocellular Department paraventrikulyarnoe kernel (figure 8, A), Central nucleus of the amygdala (figure 8) and the kernel of a single way (NTS, figure 8, C). Among these structures only NTS is a place of expression of CRF-R2 (figure 8, D). Other major sites of expression of CRF-R2, including ventromedial nucleus of the hypothalamus (figure 8, F) do not show induced urocortin II Fos expression in the investigated dose limits peptide (1-10 µg). All micrographs were made with increasing X.
The figure 9 shows the activation of Central stress-related groups of cells after Central injection related urocortin peptide through research stimulation of adenauerallee FOS in the terminal strip (stria terminalis) (BST), paraventrikulyarnoe nucleus of the hypothalamus (PVH), the Central nucleus of the amygdala (CeA), lateral parabrachial kernel (PBI), bluish ((locus coeruleus) (LC)) and the kernel of a single way (NTS). BSTov, bed nucleus stria terminalis (oval bottom core); ic, internal capsule; CP, caudate nucleus/putamen; ac, anterior commissure; V3, third ventricle; AHA, anterior region of the hypothalamus; pm, posterior magnocellular part (paraventrikulyarnoe core); fx, fornix; CeAm, the Central nucleus of the amygdala; BLA, bazo-lateral nucleus of the amygdala; scp, the upper leg of the cerebellum; PBel, parabrachial the core (outer lateral part); V4, fourth ventricle; ep, ependyma; AP, the rear field; DMX, dorsal motor nucleus of the vagus nerve; ts, single path; and cc, Central canal.
In figures 10A and 10B shows the effect of centrally injected urocortin II on food consumption and General motor activity. Figure 10A shows the average (±SEM; n=3-6 per group) total food consumption at night (g) after icv administration of 1 ág CRF, urocortin or urocortin II. CRF and urocortin significantly reduced food consumption compared to controls, which were injected with saline solution, starting from 4 h after injection, whereas the effect of urocortin II was not evident until 6 h after treatment. *p< 0,002 (CRF and Ucn in comparison with saline solution),**p< 0,002 (CRF, urocortin and urocortin II compared with saline). Figure 10B shows the telemetric measurement of total motor activity, which were significantly increased in animals that received icv injection of CRF; neither urocortin or urocortin II had no significant effect on motor activity. *p< 0,001 (CRF compared with saline solution).
The figure 11 shows the stimulation of ACTH secretion from cells of the anterior pituitary by urocortin and related urocortin peptide person. Cells in the anterior pituitary of rats was injected into the culture and treated or urocortin rats or related urocortin peptide person. Secreted ACTH was measured using a kit (Nichols Institute Diagnostics).
The figure 12 shows the impact of related urocortin peptide person on camp levels in A7R5 cells that Express native CRF-R2β. Dose-dependent effect of incubation with urocortin (light circles) or hURP (filled circles) for 30 minutes on the production of camp. camp was measured by RIA (Biochemical Technologies).
The figure 13 shows the impact of related urocortin peptide person (hURP) on General motor activity in rats.
The figure 14 shows the effect intracerebroventricular injection related urocortin peptide person (URP) on body temperature of rats.
N the figure 15 shows the effect intracerebroventricular injection related urocortin peptide person (hURP) consumption by rats of food at night.
The figure 16 shows a model of how related urocortin peptide person acts on CRF-R1 and CRF-R2. Related urocortin peptide person with high affinity binds to CRF-R2, but not CRF-R1, whereas urocortin binds to both receptors. CRF with high affinity binds to CRF-R1, but not associated with CRF-R2.
DETAILED description of the INVENTION
According to this invention it is possible to use traditional methods of molecular biology, Microbiology and recombinant DNA technology developed in this area. A full explanation of these methods is available in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis (M.J. Gait, ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames and S.J. Higgins, Eds. (1985)]; "reduced and Translation" [B.D. Hames and S.J. Higgins, Eds. (1984)]; "Animal Cell Culture" [R.I. Freshney ed. (1986)]; "Immobilized Cells and Enzymes [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).
Therefore, the following terms when they appear in this specification, shall have the definitions stated below.
In the sense used here, the term "cDNA" will refer to a DNA copy of the mRNA transcript of the gene.
In the sense used here, the term "derived amino acid sequence" will refer to the amino acid sequence determined by reading the sequence triple the s bases of the nucleotides in the cDNA.
In the sense used here, the term "screening library" will refer to the usage of labeled probe to ensure that under appropriate conditions to check whether a specific library DNA sequence complementary to the probe. In addition, screening libraries can be performed by PCR.
In the sense used here, the term "PCR" refers to the polymerase chain reaction, which is the subject of U.S. patent No. 4683195 and 4683202 Mullis, as well as other modifications known in the present time in this area.
All sequences of amino acid residues represented by formulas that location from left to right correspond to the normal direction from aminobenzo to the carboxyl end. In addition, it should be noted that a dash at the beginning or at the end of the sequence of amino acid residues indicates a peptide bond with another sequence of one or more amino acid residues.
These amino acids are preferably in the L-isomeric form. However, any residue of L-amino acids may be substituted residues in the "D"isomeric form, provided that the desired functional property of the polypeptide to contact the immunoglobulin is saved. NH2refers to the free amino group present on Amin is the end of the polypeptide. COOH refers to the free carboxyl group present on the carboxyl end of the polypeptide.
Non-standard amino acids can be included in proteins by chemical modification of existing amino acids or as the result of artificial protein synthesis. Non-standard amino acid refers to an amino acid that differs in chemical structure from the twenty standard amino acids encoded by the genetic code. Post-translational modification in vivo may also lead to the presence of non-standard amino acid or derived amino acids in the protein. N-terminal NH2- and C-terminal COOH groups of the protein can also be modified as a result of natural or artificial post-translational modification of a protein.
Proteins can be modified by substitutions of amino acids. Often some changes result in significant changes in the activity of the proteins, while others have little effect or no effect. Less likely is that conservative substitutions strongly modulate the activity of the protein. "Conservative amino acid substitution" refers to substitution of an amino acid with a chemically similar amino acid, i.e. the replacement of non-polar acid other nonpolar amino acid; replacement of a polar amino acids other polar amino acid residue other is acidic amino acids, etc. are the Examples of the preferred conservative substitutions are shown in table 1:
|The original balance||Preferred conservative substitutions||Preferred conservative substitution|
|Ala (A)||Val; Leu; Ile||Val|
|Arg (R)||Lys; Gln; Asn||Lys|
|Asn (N)||Gln; His; Lys; Arg||Gln|
|His (H)||Asn; Gln; Lys; Arg||Arg|
|Ile (I)||Leu; Val; Met; Ala; Phe; Nle||Leu|
|Leu (L)||Ile; Val; Met; Ala; Phe; Nle||Ile|
|Lys (K)||Arg; Gln; Asn||Arg|
|Met (M)||Leu; Phe; Ile||Leu|
|Phe (F)||Leu; Val; Ile; Ala||Leu|
|Tyr (Y)||Trp; Phe; Thr; Ser||Phe|
|Val (V)||Ile; Leu; Met; Phe; Ala; Nle||Leu|
"Chemical derivative" refers to a discussed polypeptide having one or more residues chemically derivatizing by the reaction of a functional side group. Such derivateservlet polypeptides include, for example, polypeptides in which free amino groups have been derivatization to form the amine hydrochloride, para-toluene sulfanilic groups, carbobenzoxy, tert-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups can be derivateservlet with the formation of salts, methyl or ethyl esters or other types of esters or hydrazides. Chemical derivatives may include peptides that contain one or more derivatives of amino acids of natural origin of the twenty standard amino acids. For example, serine is possible to replace 4-hydroxyprolin; and lysine could be replaced by ornithine. Peptides covered by this invention also include peptides having one or more connections and/or deletions of residues compared with the specific peptide is a home, the sequence of which is specified here, provided that the modified polypeptide retains the desired biological activity.
"Replicon" is any genetic element (e.g., plasmid, chromosome, virus)that functions as an Autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
"Vector" is a replicon, such as plasmid, phage or cosmid, which can be linked to another segment of DNA in order to replicate the associated segment.
"DNA molecule" refers to a polymeric form of deoxyribonucleotides (Denisovich, Gurinovich, siminovich or casinovip), either in its single-stranded form or in the form of a double helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes Dunaeva DNA found among others in the form of linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. When discussing here the structure according to the usual arrangement is the only sequence in the direction from 5' to 3' along the noncoding DNA strand (i.e., the strand having a sequence homologous to the mRNA).
"Start replication" refers to the PEFC is deuternomy DNA involved in DNA synthesis.
"Coding sequence" DNA is a double sequence of DNA that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined according to a start codon at the 5'(amino)end and a stop codon broadcast on the 3'(carboxyl) end. The coding sequence can include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequence of eukaryotic DNA (e.g. DNA mammals), and even synthetic DNA sequences. The polyadenylation signal and the sequence termination of transcription, as a rule, will be localized to the 3'side of the coding sequence.
Transcriptional and translational regulatory sequences are regulatory DNA sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for expression of the coding sequence in a cell host.
"Promoter sequence" is a regulatory region of DNA capable of binding RNA polymerase in a cell and initiate transcripts the Yu coding sequence, located to the right (3'-direction). In order to determine according to this invention, the promoter sequence is limited at its 3'-end of the site of transcription initiation and extends to the left (5'-direction), including the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be the site of transcription initiation, as well as domains, binding protein (consensus sequences)responsible for the binding of RNA polymerase. Eukaryotic promoters are often, but not always, contain "TATA"boxes and "SAT"boxes. Prokaryotic promoters in addition to the consensus sequences at -10 and -35 contain sequences Shine-Dalgarno.
"Sequence expression regulation" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. The coding sequence is "under the control" of transcriptional and translational regulatory sequences in the cell when RNA polymerase Transcriber the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
"Signal sequence" can be built near his summe is zi coding sequence. This sequence encodes a signal peptide, N-terminal relative to the polypeptide, which gives the cell master signal about the direction of the polypeptide to the cell surface or secretion of the polypeptide into the environment, and the specified signal peptide is cut the host-cell before the protein leaves the cell. The signal sequence can be detected associated with various proteins characteristic of prokaryotes and eukaryotes.
The term "oligonucleotide" is used here in the sense of a probe according to this invention, is defined as a molecule containing two or more ribonucleotides, preferably more than three. Its exact size will depend on many factors, which, in turn, depend on the ultimate function and use of the oligonucleotide.
The term "primer" in the sense used here refers to an oligonucleotide, or having a natural origin as in a purified product restriction cleavage, or obtained synthetically, which is capable of acting as a point of initiation of synthesis when it is placed in the condition in which induced the synthesis of the product of the elongation of the primer, which is complementary strands of nucleic acid, i.e. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH. PR is iMER can be either single-stranded, or donativum, and must have sufficient length to preimenovati synthesis of the desired product elongation in the presence of an inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer and the application of the method. For example, for diagnostic applications, depending on the complexity of the target sequence oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
Here the selected primers in "substantially" complementary to the different strands of a particular sequence of target DNA. This means that the primers must be substantially complementary to gibridizatsiya with their corresponding threads. Therefore, the sequence of the primer need not reflect the exact sequence of the matrix. For example, complementary nucleotide fragment may be linked to the 5'-end of the primer, with the remainder sequence of a primer complementary strands. Alternative complementary bases or longer sequences can be dispersed in the primer, provided that the sequence of the primer is sufficiently complementary sequences or hybridizes with it and thus forms a matrix for the synthesis of PR the product extension.
In the sense used here, the terms "restriction endonuclease" and "restriction enzymes" refer to enzymes, each of which cuts Dunaeva DNA at specific nucleotide sequences or near it.
The cell is "transformed" by exogenous or heterologous DNA when DNA was introduced into the cell. Transforming DNA may be integrated (covalently linked) or not integrated into the host cell genome. For example, in prokaryotes, yeast and in mammalian cells transforming DNA can be maintained in episomal element, such as a plasmid. As for eukaryotic cells, a stably transformed cell is a cell in which the transforming DNA was integrated into the chromosome, which is inherited by daughter cells through chromosome replication. On the stability demonstrated by the ability of eukaryotic cells to form lines or clones of cells, consisting of a population of daughter cells containing the transforming DNA. "Clone" is a population of cells derived from a single cell or predecessor by mitosis. "Cell line" is a clone of the primary cell, which is capable of stable growth in vitro for many generations.
Two DNA sequences "substantially homologous" in t the m case, when at least about 75% (preferably at least about 80% and most preferably at least about 90%, or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are homologous to a large extent, can be identified by comparison with a standard computer program sequences available in data banks sequences, or experiment by southern hybridization, for example, in hard conditions, which determine for a particular system. Defining appropriate hybridization conditions known to specialists in this field. See, for example, Maniatis et al., above; DNA Cloning, Vols I and II, above; Nucleic Acid Hybridization, above.
Heterologous region design DNA is an identifiable segment of DNA within a larger DNA molecule, which in nature is not detected associated with a larger molecule. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flanks the genomic DNA of mammals in the genome of the source organism. In another example, the coding sequence is a construct where the coding sequence itself is not observed in nature (e.g., cDNA, when a gene is MNA the coding sequence contains introns, or synthetic sequences having codons different from the native codons of the gene). Alellie options or mutational events of natural origin do not cause the appearance of heterologous DNA, the definition of which is here given.
Labels are normally used for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet radiation, and other marks. There are many fluorescent materials, and you can use them as labels. Such materials include, for example, fluorescein, rhodamine, auramine, Texas red, AMC blue and Lucifer yellow. Specific detection material is anti-rabbit antibody obtained in the body of a goat and conjugated with fluorescein using isothiocyanate.
Specific analysis system, developed and used in this area, known as receptor analysis. The receptor in the analysis of the material that you want to analyze, as appropriate mark, and then in some of the tested colonies of cells make for some number of labels, followed by a research associate, in order to determine the extent to which the labeled material bound to cellular receptors. Thus it is possible to establish differences between the materials on the affinity.
In the sense used here means that the term "owner" includes not only prokaryotes, but also eukaryotes, such as yeast cells, plants and animals. A recombinant DNA molecule or gene, which encodes a protein according to this invention, can be used to transform a host using any of the methods generally known to experts in this field. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeast, such as Pichia pastoris, mammalian cells and insect cells.
In General, expressing vectors containing promoter sequences which facilitate the efficient transcription of the built-DNA fragment, used in connection with the owner. Expressing the vector typically contains the beginning of replication, a promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. Transformed hosts can ferment and cultivate according to the methods known in this area for optimum cell growth.
To construct expressing vectors containing the appropriate signals in the regulation of transcription and translation, you can use the methods well known to experts in the Noi area. See, for example, the methods described in Sambrook et al., 1989, Molecular cloning: A Laboratory Manual (2ndEd.), Cold Spring Harbor Press, N.Y. Gene and sequence regulation of transcription is called "operatively connected", if the sequences regulating transcription effectively control the transcription of the gene. The vectors according to the invention include, but are not limited to, plasmid vectors and viral vectors.
The direction of this invention is the DNA sequence encoding urocortin II. This sequence can be isolated and purified DNA which encodes urocortin II. Alternative, it can represent an isolated and purified DNA, which in conditions of high stringency hybridized with antisense sequences, complementary DNA urocortin II, under conditions of high stiffness (defined as flushing the membrane at high temperature and low salt concentration, is functionally equivalent to 0.1 x SSC at 65°). Finally, DNA can be an isolated and purified DNA encoding urocortin II, but which differs in sequence due to the degeneracy of the genetic code. Specified DNA preferably encodes a protein with amino acid sequence SEQ ID NO: 10 or an amino acid sequence of SEQ ID NO: 11.
Direction this is about of the invention is a vector able to Express urocortin II. Such a vector comprises DNA that encodes urocortin II, and the regulatory elements necessary for expression of urocortin II in the cell. In a preferred embodiment, the specified vector encodes a protein with amino acid sequence SEQ ID NO: 10 or an amino acid sequence of SEQ ID NO: 11. The direction of the present invention is a host cell, transfusiona this vector and expressing urocortin II with such a vector. Protein can be Express in the type of cells selected from bacterial cells, mammalian cells, plant cells and insect cells. In a preferred embodiment, the protein is expressed in E. coli.
The direction of the present invention is isolated and purified protein of urocortin II encoded by the DNA described above. Preferably the purified urocortin II has an amino acid sequence corresponding to SEQ ID NO: 10 or SEQ ID NO: 11.
The direction of the present invention is an antibody directed against the protein of urocortin II. The specified antibody is preferably a monoclonal antibody.
In addition, the direction of the present invention is a pharmaceutical composition comprising the protein of urocortin II and a pharmaceutically acceptable carrier. Such pharmaceutical composition can be used is to use for lowering the temperature of the body, suppress appetite, treatment or prevention of congestive heart failure, treatment of stress and anxiety and change undesirable low levels of ACTH secretion.
The direction of the present invention is a DNA sequence encoding a related urocortin peptide person. This sequence may represent an isolated and purified DNA which encodes a related urocortin peptide person. Alternatively, it can represent an isolated and purified DNA, which in conditions of high stringency hybridized with antisense sequences, complementary DNA related urocortin peptide person (conditions of high stringency is defined as the flushing of the membrane at high temperature and low salt concentration, is functionally equivalent to 0.1 x SSC at 65°). Finally, DNA can be an isolated and purified DNA encoding related urocortin peptide person, but which differs in sequence due to the degeneracy of the genetic code. Specified DNA preferably will have a sequence shown in SEQ ID NO: 1 and preferably will encode the precursor protein having the amino acid sequence of SEQ ID NO: 2, which proteoliticeski processed in baie is OK with the amino acid sequence SEQ ID NO: 3.
The direction of the present invention is a vector that can Express the related urocortin peptide person. Such a vector comprises DNA that encodes a related urocortin peptide person, and the regulatory elements necessary for expression related urocortin peptide man in the cage. In a preferred embodiment, the specified vector encodes a protein with amino acid sequence SEQ ID NO: 3. The direction of the present invention is a host cell, transfusiona this vector and expressing the related urocortin peptide person with the specified vector. Protein can be expressed in a cell type selected from bacterial cells, mammalian cells, plant cells and insect cells. In a preferred embodiment, the protein is expressed in E. coli.
The direction of the present invention is isolated and purified protein related urocortin peptide person, encoded by the above DNA. Preferably cleaned related urocortin peptide person has an amino acid sequence corresponding to SEQ ID NO: 3.
The direction of the present invention is an antibody directed against a protein related urocortin peptide person. The specified antibody is preferably a monoclonal antibody.
Cu is IU, the direction of the present invention is a pharmaceutical composition comprising a protein related urocortin peptide man and a pharmaceutically acceptable carrier. Such pharmaceutical composition can be used to reduce body temperature, appetite suppressant, treatment or prevention of congestive heart failure, treatment of stress and anxiety and change undesirable low levels of ACTH secretion.
The direction of the present invention is also urocortin II or related urocortin peptide human, mutated so that it contained a tyrosine residue that is designed to radioiodine protein. One of the specific modifications is joining a sequence of Tyr-Gly, N-end-urocortin II or related urocortin peptide person.
The direction of this invention also are deletion mutants of urocortin II or related urocortin peptide person. Particularly suitable deletion is a deletion from one to five amino acids from the N-Terminus of the protein.
The direction of the present invention is also urocortin II or protein related urocortin peptide human, which is the standard isomeric L-form amino acids substituted isomeric D-forms of amino acids. In a protein related urocortin person, the item is suitable substitution of the isoleucine residue, corresponding to position 9 of SED ID NO: 3, D-isoleucine, D-phenylalanine and D-leucine or D-form of another amino acid. Other applicable replacement is the replacement of the glutamic acid residue at position 17 of SEQ ID NO: 3 or 11 D-glutamic acid.
The direction of the present invention is also urocortin II or related urocortin peptide person, in which the various amino acids have been replaced by non-standard amino acids. Examples of such non-standard amino acids areα-methylated leucine,α-methylated alanine, N-im-benzylation, 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine and ornithine.
The direction of the present invention is also urocortin II or protein related urocortin peptide person having acylated N-end. The specified protein acylation can be used to bind molecules, such as fatty acid N-end of the protein to protect Ucn II or URP from enzymatic cleavage or to change various properties of the protein, such as its hydrophilicity/hydrophobicity. The above modification can be used to change the duration or bioavailability of the protein in vivo.
The direction of the present invention is also urocortin II or protein related urocortin peptide person which has been modified so that to contain a fluorescent label, for the purpose of rendering or biological analysis.
The direction of the present invention is also urocortin II or protein related urocortin peptide person, conjugated with a complexing agent for radionuclides. Ucn II in complex with a radionuclide can be used for scintigraphy or in different tests.
The direction of the present invention is also urocortin II or related urocortin peptide (human) conjugated with a toxin. The resulting toxic conjugate can be used for targeted resolution cells bearing the receptor for CRF.
The following examples are given to illustrate different variants of the invention and in no way imply a limitation of this invention.
IDENTIFICATION of RELATED UROCORTIN PEPTIDE PERSON
During attempts to identify novel ligands CRF-R has constructed a hidden Markov model (hmm) - based alignment Clustal W known protein family CRF, including CRF rat/human, rat Ucn, Ucn person, sauvain frogs and urotensin I remoras, using the software package HMMER (Sean Eddy, Department of Genetics, Washington University, St. Louis, MO; see reference 19). Specified MMOs used to search the published database of the genome is of ne, and found YOU (Genbank, inventory No: AC005903)derived from chromosome R-4, which contained an area of 109 BP, exhibiting a significant degree of sequence homology, but which was not part of a previously identified gene. The specified area was extended up to 621 BP ID EST clone humans, which overlap with this sequence (Genbank, inventory No. BE622276). However, in the sequence a person lacks the consensus site of proteolytic cleavage, which would provide the possibility of C-terminal processing of the peptide. Therefore, the protein was identified as a sequence of related urocortin peptide person (hURP). The figure 1 shows the nucleotide sequence (SEQ ID NO: 1) designed open reading frame protein URP person. Specified gene encodes a peptide with the amino acid sequence SEQ ID NO: 2.
To confirm the existence and sequence of a gene related to urocortin peptide person, used oligonucleotide primers, similar to the primers used for amplification of sequences related urocortin peptide man from a genomic clone to isolate a partial cDNA fragment by PCR from a cDNA library of the pancreatic islets of man. This fragment was also subcloned into the vector pGEM and sequani is ovale. The cDNA sequence corresponded to a part of the genomic sequence. A partial cDNA sequence corresponds to the underlined sequence in figure 2. The sequence shown in figures 1 and 2 encode the polypeptide precursor related urocortin peptide person. The first 19 nucleotides related urocortin peptide human encode a signal peptide that is cleaved during post-translational modification of the protein, giving Mature related urocortin peptide person with the amino acid sequence:
I V L S L D V P I G L L Q I L L E Q A R A R A A R E Q A T N A T R I L A R V G H C-NH2(SEQ ID NO: 3).
The figure 3 shows the results of the comparison of homology between amino acids 72-109 related urocortin peptide of human rights and equivalent segments urocortin person, urotensin I human corticotropin-releasing factor (CRF), sauvagine frogs and CRF/Uro sea dogs. Homology in this region is in the range from 26 to 42%.
IDENTIFICATION of UROCORTIN II MOUSE
The fragmented cDNA probes derived from the gene sequence of human-specific cross was hybridisable with cloth rats (brain), suggesting that there is a reasonable degree of homology between the two species. Based on this sequence, the person designed the primers listed, to identify homologous gene of the mouse by way of rapid amplification of cDNA ends (RACE). Prepared by RACE cDNA was obtained from poly(A+)RNA of the whole brain of a mouse with the use of the kit for the amplification of cDNA RACE SMART (Clontech). The PCR reactions were carried out in conditions of low stringency (low Tmto try and ensure that heterologous priming. The first round of amplification was performed using the Protocol "touchdown" (94°C, 30 s; step from the 70°C to 55°C, 30 s; 72°C, 3 min) followed by a second round of amplification with multiple sets of nested primers (94°C, 20 s; 55°C, 20 s; 72°C, 3 min). Selected to test the PCR products were cloned into pCRII-TOPO (Invitrogen) for sequencing both strands. Selected for test 5'- and 3-the reaction products were identified on the basis of their predicted size (calculated based on a series of man), cloned and sequenced.
The calculated amino acid sequence of Ucn II mouse shown in Fig. 4A. The gene encodes the precursor of 112 amino acids, and the end contains the coding region of the proposed Mature peptide of 38 amino acids specified in the area bounded by the frame (Fig. 4A). For the C-terminal part of the coding sequence is followed by a glycine and paired basic residues (R-R), alleged the nutrient involved in the amidation and the departure from its predecessor, respectively.
There are two other suspected or known related urocortin peptide: peptide person, the peptide sequence of which is calculated on the basis of the published EST man, and the recently cloned (20) URP fish-dog (Takifugu rubripes). Alignment with related urocortin peptides of human and fish, rat Ucn and CRF rats/person shown in Fig. 4B. At the amino acid level, the coding region Ucn II mouse exhibits 77 and 45% homology with related urocortin peptides of human and fish, respectively. Ucn II mouse relatively close to known representatives of this family of peptides, showing 36 and 44% amino acid identity with CRF rats and UCN rats, respectively. Assuming a conservative replacement, the similarity increases to 62% (CRF) and 59% (Ucn).
SYNTHESIS of PEPTIDES
Ucn II and mouse Ucn-related peptide person synthesized manually using solid-phase method, a resin-based methylbenzhydrylamine and Boc-strategy (21). To remove the Boc-group was used triperoxonane acid, 60% in dichloromethane. The Assembly of the main chain was performed using diisopropylcarbodiimide. The peptides were tsalala and removing the protective group in the fluorine-hydrogen acid and was purified using RP-HPLC and three systems solvent (phosphate triethylamine at pH of 2.25 and 6.5 and/or 0.1% TFU) (22). The peptides had a purity ball is e 95% according to independent criteria HPLC and CZE. To confirm the composition of the drugs used mass spectra.
ACTIVATION of the RECEPTORS BY Ucn II
The affinity of Ucn II in relation to the receptors CRF-R1 and CRF-R2 was assessed using a radioreceptor assay. Crude membrane fraction was obtained from cells SNO, stably expressing either cloned CRF-R1 or CRF-R2β. The tested peptides and radioligand,125I-[Tyr0Glu1, Nle17]-sauvagine, diluted in buffer for analysis (20 mm HEPES, 2 mm EGTA, 0.1% BSA, 10% sucrose, pH 7.6) and was mixed with membrane preparations receptors in tablets for micrometrology MAGV (Millipore)pre-coated with 0.1% polyethylenimine. The reaction mixture was incubated for 90 min at room temperature, and then quickly washed twice with buffer for analysis and filtered. Complex radioligand quantitatively assessed through accounts of gamma radiation. The inhibition constants of binding was determined using the computer program Prism. The results are summarized in table 2.
|Binding properties and functional activity of selected ligands of receptors CRF|
|Urocortin II (mouse)||>100||>100||0,66(0,13-3,3)||of 0.14(0.03 to 0.52 in)|
|Urocortin (rat)||of 0.32(0.14 to 0,77)||0,29 (0,12-0,70)||of 0.62(0.14 to 2,8)||0,17(0,043-0,68)|
Values were determined from 3-6 independent experiments using stable transfetsirovannyh cells SNO or membrane for each of the test peptide. The values of EC50and Kiwas determined using the computer program Prism. The values of their log10were averaged (γ). Average EC50or Kiwas taken as equal to 10γ. Expected standard deviation of the values log10(δ). Limits were taken as equal to [(10γ)10δor 10γ/10δ].
Compared with urocortin Ucn II was at least 1000 times less effective at competing with labeled sauvagine for binding to CRF-R1, whereas it is about the agave almost equal efficiency with Ucn when competing for binding to CRF-R2. Found significant selectivity against receptor type 2 is also observed in case of activation of the receptor, which was measured by the accumulation of intracellular camp. Stable transfetsirovannyh cells Cho (cultured in DMEM/10% FBS) were sown in a 48-hole Cup for tissue culture (Costar) and allowed to recover for 24 hours. The medium was replaced with DMEM/0.1% of FBS for at least two hours before processing. The cells were pre-incubated for 30 min with 0.1 mm 3-isobutyl-1-methylxanthine and then were exposed to the effect of peptides for 20 min at 370C. were Extracted intracellular camp and was measured in two repeats from the wells three times using the set for RIA (Biomedical Technologies). In the analysis of camp Ucn II showed comparable with Ucn effectiveness against CRF-R2 (table 2). Extremely low affinity Ucn II in respect of CRF-R1 interfere with the determination of its efficiency at this receptor.
Binding was performed in 96-well tablets Durapore, 0.2 μm, using a filtration system for multiple screening assay (Millipore). Each well contained a total volume of 200 μl consisting of 50 μl of buffer for binding (10% sucrose, 0.1% BSA, 2 mm EGTA, 20 mm HEPES buffer, pH 7.5); 50 μl of unlabeled competing agent (urocortin or related urocortin peptide person) at different intelligence is the third in the buffer for binding; 50 μl of125I sauvagine concentration 150000 counts/min/well; 50 μl of cell membranes. The plates were incubated for 1 hour at room temperature, was filtered in vacuum, washed twice with a buffer for binding and allowed to dry. Individual filters were cleared and counted using a gamma counter.
Indirectly related urocortin peptide person substitution125I sauvagine, communicating with CRFR1 and CRFR2β, stably expressed in cells SNO shown in figures 5A and 5B. Based on the data found that related urocortin peptide person has a dissociation constant (Ki), equal to 78 nm for CRF-R1 and 0.23 nm for CRF-R2β. On the other hand, Ucn had a dissociation constant equal to 0.13 nm for CRF-R1 and 0.15 nm for CRF-R2β. Therefore, related urocortin peptide person much more specific receptor for corticotropin-releasing factor type II than urocortin.
EXPRESSION of mRNA Ucn II
Histochemical analysis of the hybridization was performed to analyze the expression pattern of mRNA Ucn II in the brain of mice and rats. Animals were subjected to deep anesthesia with chloral hydrate (350 mg/kg/b) and perfesional through the ascending part of the aorta arch saline solution, and then ice-cold 4% paraformaldehyde in 0.1% borate buffer, R is a 9.5. The brain was postvaccinial for 16 hours and subjected to cryogenic freezing overnight in 10% sucrose in 0.1 M phosphate buffer. Cut four (mouse) or six (rats) series frozen sections with a thickness of 30 μm using a sliding microtome, collected in a cold cryoconservation based on ethylene glycol and stored at -20°to histochemical processing.
The in situ hybridization was performed using35S-labeled antisense and sense (control) probes crnc (23), first designed by linearization of the plasmid TOPO-II, containing the cDNA of the mouse. Probes were labeled to a specific activity of 1-3 x 109location./min/µg, were applied to the slides at a concentration of about 107pulse/min/ml and hybridized overnight at 56°in conditions of high stringency (50% formamide). Final washing was performed in 15 mm NaCl/1.5 mm sodium citrate at 65-68°C. slides were then obezvozhivani and exposed to x-ray film (βMax; Kodak) for 16 h, and then was coated with liquid emulsion (Kodak NTB-2 and exhibited at 4°within 21-28 days.
Histochemical analysis of hybridization showed compatible and limited expression of mRNA Ucn II in the brain of mice and rats. Probes based on the semantic threads marked to similar specific activities, as antisense probes, would not exceed the surrounding background hybridization signals. Registered mRNA Ucn II was mainly observed in subcortical areas, with the main places of expression included stress-related groups of cells, such as paraventrikulyarnoe, supraopticus and the arcuate nucleus of the hypothalamus, and blue place (locus coeruleus) Rostral sections of the bridge (Fig. 6). Motor nuclei of the brain stem (trigeminal, facial, hypoglossal nerves), and ventral horn of the spinal cord also identified as sites of mRNA expression Ucn II. Among negarandeh elements of positive hybridization signals were observed uniformly in the membranes of the brain, but not in chorioidea plexus or ependyma. Not received evidence of expression of mRNA Ucn II glial elements.
The EXPRESSION of RELATED UROCORTIN PEPTIDE IN the BRAIN of PRIMATES
The expression of related urocortin peptide of human rights in the brain of primates investigated by in situ hybridization. The in situ hybridization was performed on sections of brain tissue Macaca fascicularis using35S-labeled antisense probe crnc corresponding to approximately 400 base pairs related urocortin peptide person. The probe was applied to the slides at a concentration of 107pulse/min/ml, and gave the opportunity for hybridization to continue throughout the night. After hybridization pre is a noticeable glass was treated with 20 μg/ml of ribonuclease And for 30 minutes at 37° C and washed with 15 nm NaCl/1.5 mm sodium citrate/50% formamide at 70°C. Cover glasses were obezvozhivani and exposed to x-ray film (BetaMax; Kodak) for 24 hours. Autoradiogram sample shown in Fig. 7. A positive signal for URP observed in paraventrikulyarnoe (PVH) and supraopticus nuclei of the hypothalamus primates.
The EXPRESSION of Fos INDUCED by Ucn II
To identify groups of cells that meet at a Central introduction Ucn II, and to estimate corresponding to these cells spread in places the expression of CRF-R2, monitored induced expression of the gene product of immediate and early response Fos in response to icv-injection of peptide. Before the experiment, adult male rats Sprague-Dawley (250-300 g at the beginning of the experiment) and mice S BL/6 (25-40 g) contained in the premises for breeding cycle light:dark 12:12 and with free access to food and water. For intracerebroventricularly (icv) injection, rats were anestesiologi ketamine/xylazine/acepromazine, and stereotaxically implanted them guide cannula gauge 26 terminating in lateral ventricle. For intravenous (IV) injection of peptides in the jugular vein of the animals were injected with permanent catheters. Rats treated with icv injection, also intraperitoneally implanted sensor at a distance to control the levels of the total active is minute and body temperature (Mini-Mitter). After surgery, animals were given an opportunity to recover for 7 days before any of the experiments, and within the specified period, the animals were daily taken in hand. All procedures were approved by the institutional Committee on the content and use of animals Salk Institute.
To trace the characters induced Fos expression in rats at 10 a.m. or icv, or in/were injected with synthetic Ucn II (1, 5 or 10 μg/animal in 2 μl saline icv injection, or 200 ál for/in the introduction), or only one filler, and perfesional two hours later. To trace the impact of the introduction of the peptide on food intake in animals conducted icv-injection of synthetic Ucn II of mouse, rat Ucn or CRF rats/person for 30 minutes before turning off the light. Then measured the consumption every hour for 6 h and 12 h of Data were analyzed using analysis of variance of repeated measurements (ANOVA) with amendments Bonferroni for multiple comparisons applied when it is justified.
For immunohistochemical analysis of tissue pre-consistently treated with 0.3% hydrogen peroxide and 1% sodium borohydride. Then permeabilization in PBS/0.2% Triton X-100 and incubated with the first anticorodal within 48 h in PBS/2% blocking serum. Fos immunoreactivity was localized using polyclonal Inoi antisera, obtained in rabbits against the N-terminal synthetic protein fragment Fos man (Santa Cruz Biotechnology, 1:5K). Localization was performed using traditional immunoperoxidase way with Avidya-Biotin and strengthening of Nickel, as described in (24).
Injection of 1 μg of synthetic Ucn II evoked activation responses, which the most notable were in a group of interconnected structures involved in Central autonomic regulation (25, 26). These patterns included a separate provision in the bed nucleus of the terminal strips (the stria terminalis), the Central nucleus of the amygdala, paraventrikulyarnoe nucleus of the hypothalamus (PVH), parabrachial the nucleus and the nucleus of the solitary path (NTS; Fig. 8). Only NTS has been described as the locus of expression of CRF-R2 (27). Induction of Fos in the other main sites of expression of CRF-R2, including the lateral septum, nucleus seam mid-brain and ventromedial nucleus of the hypothalamus (27, 28), did not differ from induction observed in controls with injections of saline. Higher doses of peptide (5 or 10 μg) evoked stronger activation responses similar distribution.
In order to control the potential systemic effects of icv-injection of a similar dose limits Ucn II was injected intravenously separate groups of rats. Only the highest dose (10 µg) caused induction of Fos, which was significantly higher counter is selected levels. Although the pattern was similar to that observed in response to Central injection or the number of labeled cells or the intensity of staining was not reached that level, which is reliably observed after icv injection of 1 μg Ucn II.
The EXPRESSION of FOS IN the BRAIN, STIMULATED RELATED UROCORTIN PEPTIDE
The activation of Central stress-related groups of cells related urocortin peptide person has been evaluated by identifying gene product Fos in cells after injection related urocortin peptide person. Rats implanted guide cannula into the lateral ventricle of the brain, seven days before the experiment. On the day of testing, rats were injected with 5 μg of synthetic related urocortin peptide person in 5 μl of sterile saline. Rats were scored in two hours, and was prepared by coating flowed from the different sections of the brain. Cover glasses were stained for localization of Fos immunoreactivity immunoperoxidase method using polyclonal serum obtained from rabbits against residues 3-16 protein Fos person.
As shown in figure 9, Fos immunoreactivity was found in the bed nucleus of the terminal strips (BST), paraventrikulyarnoe nucleus of the hypothalamus (PVH), the Central nucleus of the amygdala (CeA), the lateral pairs is brachiale kernel (PBI), blue (LC) and the nucleus of the solitary path (NTS). Each of these places previously considered the place of activity of CRF related peptide.
The EFFECT of Ucn II ON BEHAVIOR
Like CRF and Ucn, Ucn II can also have an effect on the Central nervous system, inhibiting food intake (Fig. 10A). Measurements for separate groups of rats, which were injected with the indicated peptides (1 µg, icv) at the beginning of the night phase of their cycle day-night show significant interaction between treatment and time-point [F (18,95)=4,22, p<0,0001], both the main effect reached significant values. All three peptide significantly reduced food consumption during the 12-hour interval, the degree of suppression was within 30 (CRF) to 35% (Ucn II) to 70% (Ucn). These effects tended to differential distribution over time, as Ucn and CRF-treated animals ate significantly less than the control animals treated with saline, in the earlier period of the test period (4-5 hours)than rats treated with Ucn II (6 hours).
At the same specified subjects of General motor activity and body temperature was controlled by telemetry (Fig. 10V). Data analysis activity revealed a significant relationship between the action of the drug and time point [ (33,110)=1,94, p<0,006], both the main effect reached significant values. A posteriori (post-hoc comparison showed that the animals that received CRF were significantly more active than rats treated with media during the interval 2-6 h after injection (p< 0,001). No processing Ucn no processing Ucn II did not cause significant change to the specified criterion in any time point after injection. Also register the internal temperature of the body, each peptide caused relatively moderate (0.5 to 1° (C) and time (2 h) hypothermic responses (data not shown).
BIOLOGICAL ANALYSIS of MEDIATED hURP EFFECTS ON CELLS of the ANTERIOR PITUITARY gland of RATS IN VITRO
For evaluating the effect on the pituitary gland was measured response in the form of secretion of ACTH on related urocortin peptide of human rights in primary cultures of cells in the anterior pituitary of rats, as described in (30). The levels of ACTH were determined using a kit for immunoassay ACTH Nichols Institute Diagnostics. Cells in the anterior pituitary of rats were treated either with urocortin rats or related urocortin peptide person, and measured the level of secreted ACTH, using a kit (Nichols Institute Diagnostics). The influence of urocortin and related urocortin peptide person on the secretion of ACTH is shown in figure 11. Found that stimulation of the secretion of ACTH Vlatko the anterior pituitary is less sensitive to sibling urocortin the peptide person, than urocortin.
The BIOANALYSIS of INFLUENCE hURP CELLS 7R5 IN VITRO
Determined the impact of hURP in camp levels in cells 7R5 that Express native CRF-R2β. Cell line 7R5 maintained in DMEM with addition of 10% fetal bovine serum, 2 mm L-glutamine, 100 µg/ml streptomycin. Cells were sown at a density of 10,000 cells/cm2and were grown for six days. Cells deprived of serum pre-incubated with 0.1 mm 3-isobutyl-1-methylxanthine for analysis within 20 minutes and was treated with the indicated concentrations of peptide for 30 minutes. The camp levels measured by RIA (Biochemical Technologies) and is shown in figure 12. Related urocortin peptide person had a similar urocortin effect on the production of camp.
IMPACT RELATED UROCORTIN PEPTIDE MAN ON the TOTAL ACTIVITY
To determine is whether related urocortin peptide human role in the generation of the stress response, we examined the influence of related urocortin peptide person on General motor activity. Cannula surgically injected into the right lateral ventricle, whereas telemetry device implanted intraperitoneally to provide continuous monitoring of General motor activity. Animals were given a recovery period after surgery, p is wny seven days. At this time, animals were daily taken by hands, the animals were habituated to the injection procedure. On the day of injection, the initial activity was recorded for four hours. At 6:00 PM, during the dark period, animals received an injection of either 5 μl of saline or 5 µl of physiological solution containing only 5 mg related urocortin peptide person. Account activity summed over a period of time equal to four hours. The results are summarized in figure 13. Did not observe significant differences in total motor activity in animals that were injected with related urocortin peptide person, compared to control animals.
IMPACT RELATED UROCORTIN PEPTIDE PERSON ON BODY TEMPERATURE
Investigated the influence of the related urocortin peptide person on body temperature in rats. Cannula for injection related urocortin peptide man surgically injected into the right lateral ventricle. A telemetry device for continuous subtle analyses of body temperature implanted intraperitoneally. Animals were given a recovery period after surgery, is seven days. At this time, animals were daily taken by hands, the animals were habituated to the injection procedure. On the day of injection, the initial temperature of the region who were tarawali for three hours. At 6:00 p.m. (start off light), the animals received an injection of either 5 μl of saline or 5 µl related urocortin peptide person in physiological solution at a concentration of 1 µg/µl. Body temperature was monitored every five minutes for up to twelve hours. As can be seen in figure 14, the animals, which were injected with related urocortin peptide man, had a lower body temperature immediately after seven hours after the injection.
IMPACT RELATED UROCORTIN PEPTIDE PERSON ON APPETITE
Also investigated the influence of the related urocortin peptide person on appetite in rats. Cannula for injection related urocortin peptide man surgically injected into the right lateral ventricle and the animal was allowed to recover for seven days. At this time, animals were daily taken by hands, the animals were habituated to the injection procedure. On the day of injection, the animals received an injection of either 5 μl of saline or 5 µl related urocortin peptide person in physiological solution at a concentration of 1 µg/µl. The amount eaten by each animal foods were recorded every hour for six hours and at the fourteenth hour.
The total amount of food consumed during the experiments is shown for each period on figure 15A. Animals that were injected with related urocortin peptide man ate significantly less food than control animals. Figure 15 summarizes the amount of food consumed in each period of time. Animals treated hURP, much less ate food during the first and third hour after injection, and during the last eight hours of the experiment.
SUITABLE MODIFICATIONS AND DERIVATIVES of UROCORTIN II AND RELATED UROCORTIN PEPTIDE PERSON
Declared here urocortin II and related urocortin peptide person most likely to represent the form of the prohormone of these proteins. It is assumed that the activation of hormones is involved in proteolytic processing of another type of modification of proteins, such as modification, which results in pamidronate form proteins.
Previous studies of ligands for other receptors CRF showed that these ligands can be done a number of amino acid substitutions without losing the ability to bind with the receptor biological activity of the ligands. A number of previous studies urocortin showed that undoubtedly a valid one, two or even three substitutions. In some cases, modification of urocortin the result has led to a protein with more desirable pharmacological properties. As urocor is in II and related urocortin peptide is a small protein, such modification is easiest to introduce the methods of peptide synthesis are well known to specialists in this field. These methods include a method of solid-phase synthesis, partial solid phase synthesis, the condensation of fragments and classical binding in solution. These methods are particularly preferred in the case, if you want to include non-standard amino acids in urocortin II or related urocortin peptide person. Alternatively, if the modification is completely composed of natural amino acids, you can use the technology of recombinant DNA for mutagenesis and subsequent expression of the modified urocortin II and related urocortin peptide person.
In related urocortin the peptide person lacks the tyrosine residue. As the remnants of tyrosine used for radioiodine proteins, one of the possible modifications related urocortin peptide person may be replaced by another amino acid tyrosine in the protein. The previously described connection sequence consisting of Tyr-Gly into the N end of urocortin. The resulting protein retains the ability to bind to the CRF receptor and bioactivity, however, may be suitable for radioiodine protein. You can also construct other lengthening the N-Terminus of the protein according to the invention for marking other purposes.
Found that deletion of the first seven to ten residues urocortin leads to the formation of effective antagonists urocortin. These proteins are able to bind with receptors CRF, but largely do not promote or do not activate the receptors. It is assumed that the deletion of five amino acids from urocortin II or related urocortin peptide can also result in effective antagonists. You can also create antagonists from other fragments urocortin II and related urocortin peptide person. These antagonists may be effective in increasing the levels of endogenous peptides in normal clearance which is a protein that binds CRF. When linking with CRF-binding protein and blocking the binding of CRF, urocortin, urocortin II and related urocortin peptide person with the same effective protein concentration of endogenous CRF, Ucn and Ucn II increase in vivo. Such antagonists can be entered together with other agonists or antagonists of CRF, Ucn, Ucn II and URP to enhance their effect.
A comprehensive analysis of other proteins that bind to the CRF receptor, showed that replacement of the normal amino acids D-isomers of amino acids or cyklinowanie amino acids results in increased affinity relative to CRF receptors. The company is and, particularly suitable replacement is the substitution of the isoleucine residue corresponding to position 9 of SEQ ID NO: 3 or SEQ ID NO: 11, isomeric D-form amino acid, preferably D-isoleucine, D-phenylalanine and D-leucine. Similarly, the glutamic acid residue corresponding to position 17 of SEQ ID NO: 3 or SEQ ID NO: 11, you can replace the D-glutamic acid. Cyklinowanie amino acids can be formed by chemical bonds between the side chains of two or more residues. For example, nearby residues of glutamic acid and lysine can react with the formation of amide linkages, giving the ring lactam. Replacement of non-standard amino acids, such asα-methylated leucine,α-methylated alanine, N-im-benzylation, 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine and ornithine, can also be used to create agonists or antagonists related urocortin peptide person.
Stated here modification of urocortin II and related urocortin peptide person intended to illustrate possible modifications that can be done, and does not constitute a limitation of the invention.
Search homology across the genome were used to identify new members of the neuropeptides Samast is and CRF. One of the new ligands, Ucn II, selectively binds to CRF-R2 is expressed in discrete regions of the CNS of rats and activates Central neurons involved in the processing of visceral sensory information and in the modulation of the Autonomous flow. In addition, Ucn II inhibits food intake without any effect on General motor activity.
In addition to the murine peptide that exhibits structural characteristics, characteristics of the binding activity and expression expected for a representative family of CRF identified URP person (based on available published sequences EST), which is 80% identical to the sequence of the mouse at the level of nucleotides. However, explicit important difference peptide man consists in the absence of any visible site of proteolytic cleavage, which would provide C-terminal processing of human homologue. There remains a need in determining whether and how to create any homologous peptide person of a given protein. However, whereas Ucn binds with high affinity to both receptors, CRF-R1 and CRF-R2, and effectively transmits the signals through these receptors (14-16), Ucn II mouse and URP person exhibit a high degree of selectivity to CRF-R2 on the specified criteria, and will undoubtedly be set for separate functions, mediated by the two types of receptors. The figure 16 shows a model of how urocortin II effect on CRF-R1 and CRF-R2. Ucn II with high affinity binds to CRF-R2, but not CRF-R1. Urocortin binds to both receptors, whereas CRF binds with high affinity to CRF-R1, but not CRF-R2.
Ucn mRNA II demonstrates limited distribution in the subcortex in the brain of rodents, which is unique, despite the apparent partial overlap with the distribution of CRF (paraventrikulyarnoe the core; for example, reference 31) and Ucn (the brain stem and motor nuclei of the spinal cord; e.g., reference 18). Of particular interest is the fact that the transcript is expressed in a group of cells involved in stress-related physiological and behavioral functions (see reference 13). The group includes the blue place (locus coeruleus), which has extensive projections to the cortex and is involved in the creation of levels of arousal and anxiety (e.g., 32), paraventrikulyarnoe the core of which contains multiple populations of important neurosecretory neurons, and which has a projection in the Central nervous system, modulating sensory and motor flux in the Central Autonomous circuit (e.g., 33), and arcuate nucleus, which is described as the main component of the broader system, contributing to the regulation of food intake and energy balance (e.g., 34). Ho is I have no anatomical and functional data for to determine the location of new peptides in these contexts, the Central system Ucn II has the opportunity to be involved in stress-related functions, long considered the competence of the wider Central network CRF. The opposite situation exists for Ucn, principal place of expression in which the brain cells, the nucleus of Edinger-Westphal, has a very limited capacity in this regard, largely due to the small number of documented projections in the forebrain (16, 18).
From the point of view of binding parameters and the activity of the inability of the picture cell activation induced Central Ucn II, close to simulate the distribution of CRF-R2 was unexpected. A recent study, which compared the distribution of Fos expression induced by icv-introduction CRF or UCN, documentary confirmed that the pattern of activation correspond roughly to affinely binding of these peptides CRF-R encoded by two genes (35). That is, the CRF in doses similar to those used here-dose activated places the expression of CRF-R1 with a high advantage, whereas UCN caused the induction of Fos mainly in sub-groups of cells that Express each receptor. However, both peptides, in addition, involved the same set of Central Autonomous entities, to the activities identified in this work, primary space induced by Ucn II activation responses in the rat brain. It is important for the reason that parts of the Central Autonomous systems are among the best documented of places where you can act CRF-like peptides, causing stress-related autonomic and behavioral responses. These data probably indicate that activation of the receptor type 2 as type 1, is able to engage the system, although the basis for this is unknown. Among the main points in the Central autonomic network only parabrachial the core (R1) and NTS (R2) identified as designated in the expression of CRF-R (27, 28, 35), and has yet to determine whether receptor-mediated activation of one or both of these points in order to involve the whole system. It is important to note that systemic injections of synthetic Ucn II did not cause a relatively strong activation responses in the Central group of Autonomous cells in the same range of doses that are used for research in the case of icv injection. This is an important element of control, as activation of peripheral CRF-R2 can provide visible and sustained reduction in blood pressure (16, 17), and causing significant hypotension able to activate the same Central Autonomous entities that respond to the Central introduction Ucn II (36, 37).
Initial characterization of the effects of icv-entered Ucn II on food intake and activity complements recent attempts to resolve the question about the individual role of individual CRF-R associated with stress behavior. For example, while mice carrying a null mutation of one of the two receptors show a normal base level of food consumption, it is shown that animals deficient in CRF-R1, immune to anorexically effects of UCN during the period immediately after injection, but not at later time points, whereas for mice mutant for CRF-R2 opposite is true (11, 38, 39). This was seen as evidence that the early and late phases of Ucn-mediated suppression of food intake can be a event, mediated respectively CRF-R1 and CRF-R2. Using a different principle (free feeding at night, and not induced by food deprivation overeating), received data that supports this analytical conclusion as R2-specific ligand was not significantly suppressed food intake in early time points, but was suppressed later than 6 h after injection.
Measurement of motor activity also confirmed the separation of CRF-R with participation in this parameter. In accordance with the recent data on knocked-out mice, indicating that locomotor activation of t is aetsa event mediated by CRF-R1 (40), it was found that selective regarding R1 agonist, CRF, significantly increased the overall motor activity, whereas the introduction of UCN II did not cause the increase. Interestingly, treatment of UCN with high affinity binds to both receptors, resulted in a slight deviation in the direction of increased activity, while the values were significantly lower than values observed in response to CRF. This is roughly consistent with a growing body of evidence confirming the functional antagonism between the two known types of receptors. Whereas CRF-R1-deficient mice show reduced endocrine responses and answers similar to the alarming state of stress (41), the lines mutant for CRF-R2, demonstrate the increase of these parameters (11, 39, 42), suggesting that the primary activation of CRF-R2 may play a role in dealing with stress responses, managed CRF-R1.
Identification of endogenous election with respect to CRF-R2 ligand will allow us to analyse in more detail the role of the individual CRF related signaling molecules in stress-related physiological and behavioral functions. The Central expression of mRNA Ucn II coincided with the groups of cells that respond to Central injection of peptide, and confirmed behavioral responses, which according to what were previously put forward a hypothesis about the consequences of activation of CRF-R2. Further clarification of the place of the specified peptide in the biology of stress will require the identification of the Central projections of cells containing Ucn II and to identify the factors and conditions that regulate gene expression and release of the peptide.
In this description and cited the following references.
Any patents or publications mentioned in this description, indicative of levels of those skilled in the field to which this invention relates. These patents and publications is incorporated herein by reference to the same extent they would have been included if it was specifically and individually indicated that each individual publication incorporated by reference.
The person skilled in the art will readily understand that this invention is well adapted to carry out the objectives and obtain the results and advantages mentioned, as well as characteristic of the invention. The examples described here along with the methods, procedures, treatments, molecules, and specific compounds currently are examples of preferred options are illustrative and do not constitute limitations of the framework of the invention. Specialists in this field can make changes in the invention and other applications that are SOS is avnoj part of the invention, a certain part of the claims.
1. DNA encoding related urocortin peptide person where the specified DNA selected from the group including:
(a) isolated and purified DNA which encodes a related urocortin peptide person with the sequence SEQ ID No: 2-4;
(b) isolated and purified DNA that's hybrid in harsh environments with antisense sequence complementary to the selected DNA specified above in (a), where harsh conditions are defined by flushing the membrane at high temperature and low salt concentration that is functionally equivalent to 0.1 × SSC at 65°where the specified DNA encodes related urocortin peptide person with the sequence SEQ ID No: 2-4; and
(c) isolated and purified DNA with different sequences of codons from above in (a) and (b) selected DNA as a result of degeneracy of the genetic code, and which encodes a related urocortin peptide person with the sequence SEQ ID No: 2-4.
2. The DNA according to claim 1, where the sequence specified DNA represented by SEQ ID NO: 1.
3. The DNA according to claim 1, where the specified DNA encodes related urocortin peptide person with the amino acid sequence SEQ ID No: 2.
4. The DNA according to claim 1, where the specified DNA encodes related urocortin peptide person with amino acid consistently is part of SEQ ID No: 3.
5. The DNA according to claim 1, where the specified DNA encodes related urocortin peptide person with the amino acid sequence SEQ ID No: 4.
6. The vector is able to Express the DNA according to claim 1, where the specified vector contains the specified DNA and the regulatory elements necessary for expression of the indicated DNA in the cell.
7. The vector according to claim 6, where this DNA encodes related urocortin peptide person with the amino acid sequence SEQ ID No:3.
8. The vector according to claim 6, where this DNA encodes related urocortin peptide person with the amino acid sequence SEQ ID No:4.
9. The vector according to claim 6, which trapezious cells of the host.
10. The vector according to claim 9, where the specified peptide corresponds to the amino acid sequence of SEQ ID No:3.
11. The vector according to claim 9, where the specified peptide corresponds to the amino acid sequence of SEQ ID No:4.
12. The vector according to claim 9, where specified a host cell selected from the group comprising bacterial cells, mammalian cells, plant cells and insect cells.
13. The vector according to item 12, where specified bacterial cell is E. coli.
14. Isolated and purified related urocortin peptide human, selected from the group including:
(a) related urocortin peptide person with the sequence SEQ ID No:2-4 and
(b) related urocortin peptide person coded the NC, selected from the group including:
(i) an isolated and purified DNA which encodes a related urocortin peptide person with the sequence SEQ ID No:2-4;
(ii) an isolated and purified DNA that's hybrid in harsh environments with antisense sequence complementary to the selected DNA above in (i), where harsh conditions are defined by flushing the membrane at high temperature and low salt concentration that is functionally equivalent to 0.1 × SSC at 65°where the specified DNA encodes related urocortin peptide person with the sequence SEQ ID No:2-4; and
(iii) isolated and purified DNA that is different from a sequence of codons from above in (i) and (ii) the selected DNA due to the degeneracy of the genetic code, and which encodes a related urocortin peptide person with the sequence SEQ ID No:2-4.
15. Related urocortin peptide person under 14, where the specified peptide allerban fatty acid and therefore changed the time of the action or the bioavailability of the protein in vivo.
16. Related urocortin peptide person under 14, where the specified peptide modified by the introduction of fluorescent labels and therefore can be used to render the image, or biological analyses.
17. Isolated and purified related urocortin peptide person pop with the amino acid sequence SEQ ID NO:3.
18. Isolated and purified related urocortin peptide man with 14 amino acid sequence of SEQ ID NO:4.
19. The peptide according to 14, where the specified peptide amitirova-end.
20. The peptide according to claim 19, where the amino acid sequence of the indicated peptide corresponds to SEQ ID No:3.
21. The peptide according to claim 19, where the amino acid sequence of the indicated peptide corresponds to SEQ ID No:4.
22. Antibody directed against related urocortin peptide person under 14, where the specified antibody was produced using the related urocortin peptide person under 14.
23. The antibody according to item 22, where the aforementioned antibody is a monoclonal antibody.
24. Pharmaceutical composition containing related urocortin peptide person under 14 and a pharmaceutically acceptable carrier, for treating the pathophysiological condition is selected from the group including increased body temperature, appetite disorder, congestive heart failure, stress, anxiety and undesirable low levels of ACTH secretion.
25. The pharmaceutical composition according to paragraph 24, where the amino acid sequence specified related urocortin peptide person corresponds to SEQ ID No:4.
26. The modified protein, where this protein is related to urocortin peptide man on 14 and where this option is Katsia selected from the group including:
(a) adding a sequence of Tyr-Gly, N-the end of the specified protein;
(b) N-terminal deletions, where this deletion refers to the deletion of amino acids selected from the group comprising the first amino acid, the first and second amino acids, amino acids from the first to the third amino acid from the first to the fourth amino acid from the first to the fifth;
(c) replacement isolating residue corresponding to position 9 of SEQ ID No:3 or SEQ ID No:4, amino acid with a D-isomeric form;
(d) substitution of glutamic acid corresponding to position 17 of SEQ ID No:3 or SEQ ID No:4, D-glutamic acid and
(e) the acylation of N-end of a specified protein, essentially, with activity related to urocortin peptide person.
27. Protein on p, where amino acid sequence specified related urocortin peptide person corresponds to SEQ ID No:3.
28. Protein on p, where amino acid sequence specified related urocortin peptide person corresponds to SEQ ID No:4.
29. Protein on p where specified mutated protein and contains the tyrosine residue.
30. Protein on p, where the amino acid with the specified D-isomeric form selected from the group comprising D-isoleucine, D-phenylalanine and D-leucine.
31. The conjugate of the peptide for 14 or protein on p and toxin for directional RA the disruption of cells, bearing the CRF receptor.
32. The conjugate of the peptide for 14 or protein on p and complexing reagent for radionuclides.
33. Conjugate on p, in complex with a radionuclide that can be used to scintigraphie or other analyses or for the targeted destruction of cells bearing the receptor for CRF.
SUBSTANCE: method involves determining complex formed between HCV anticore antigen and NS3/4a-antibody capable of recognizing both HCV- antigens and antibodies available in sample when using common solid base and solid substrates usable in immunoassay.
EFFECT: high accuracy and sensitivity of hepatitis C diagnosis.
47 cl, 8 dwg, 10 tbl
FIELD: biotechnology, molecular biology, medicine.
SUBSTANCE: invention discloses amino acid sequences of human obesity polypeptide (OB) two isoforms possessing capacity for modulation of animal body mass, their signal peptide-containing precursors and analogues. Polypeptide isoforms are prepared as result of insertions, deletions and amino acid changes that retain activity typical for nonmodified forms of OB-polypeptides, and polyclonal and monoclonal antibodies interaction specifically with new agents modulating the body mass value also. Invention describes DNA sequences encoding these polypeptides and their analogues, vector structures comprising these sequences used for preparing recombinant forms of OB-polypeptides. Invention proposes using new polypeptides and their analogues as an active component in pharmaceutical compositions. Using this invention can promote to solving the problem for providing medicine, veterinary science and animal husbandry with effective agent used for decreasing the body mass value. Invention can be used in medicine for diagnosis and treatment of pathological states associated with disturbance of regulation of human body mass, and in animal husbandry and veterinary science.
EFFECT: valuable biological, medicinal and veterinary properties of polypeptide.
23 cl, 71 dwg, 12 tbl, 17 ex
FIELD: biotechnology, peptides, genetic engineering.
SUBSTANCE: invention relates to constructing nucleic acid molecule comprising functional in starch-containing plant tissues promoter, fragment encoding transit peptide for translocation of useful peptide into amyloplast, fragment encoding useful peptide, region encapsulating into starch and terminator. In insertion into plant genome DNA molecule expresses hybrid polypeptide comprising the desirable protein encapsulated into starch matrix. Prepared vegetable material can be used, for example, in manufacturing fodders for mammals, fishes and poultries. Also, invention can be used in food industry.
EFFECT: valuable properties of nucleic acid and polypeptides.
15 cl, 19 dwg, 9 tbl, 7 ex
FIELD: genetic engineering, pharmaceutical and medical-biological industry.
SUBSTANCE: invention proposes a chimeric sequence of nucleic acid encoding a fused polypeptide able to bind with the vessel endothelium growth factor (VEGF) and to inhibit its specific mitogenic effect. The fused polypeptide molecule comprises immunoglobulin-like domain 2 of VEGF-receptor Flt1, immunoglobulin-like domain 3 of VEGF-receptor Flk 1 or Flt4 and multimerizing component represented by either domain Fc IgG or heave chain IgG. By expression of the proposed chimeric sequence or its two successively joined copies in a host-cell a monomer or dimer of the fused polypeptide are prepared, respectively, that can be used for suppression of VEGF activity in mammals, in particular, in humans. New VEGF inhibitors differ from the known one by the improved pharmacokinetics.
EFFECT: improved preparing method, valuable biological properties of polypeptide.
23 cl, 67 dwg, 1 tbl, 35 ex
FIELD: biotechnology, microbiology, genetic engineering.
SUBSTANCE: invention proposes a new recombinant plasmid pR752 (5269 pair bases) comprising genetic construction under control of bacteriophage T5 promoter and encoding a module polypeptide consisting of 6 histidine residues, hemoglobin-like protein of E. coli, modified fragment of large T-antigen SV-40, translocation domain of diphtheria toxin, spacer sequence (Gly-Ser)5 and human epidermal growth factor (6 His-HMP-NLS-Dtox-(Gly-Ser)5-EGF) and designated for the directed transfer of photosensitizers into target-cell nuclei. By transformation of the strain E. coli M15 (rep 4) with plasmid pR752 the recombinant strain E. coli VKPM B-8356 as a producer of new polypeptide vector is prepared. The usage of this new strain is able to enhance the effectiveness of effect of photosensitizers by some orders. Invention can be used in medicinal-biological industry in preparing agents providing the directed transport of photosensitizing agents into tumor cell nuclei.
EFFECT: valuable biological and medicinal properties of polypeptide.
3 dwg, 4 ex
FIELD: medicine, oncology, biochemistry.
SUBSTANCE: invention relates to fused proteins, namely to the multifunctional fused protein cytokine-antibody. This fused protein involves immunoglobulin region and cytokine fused protein of the formula IL-12-X or X-IL-12 wherein interleukin-12 (IL-12) represents the first cytokine and X represents the second cytokine taken among the group comprising IL-2, IL-4 and GM-CSF bound covalently either by amino-end or carboxyl-end to subunit p35 or p40 of interleukin-12 (IL-12) in its heterodimeric or a single-chain form. Indicated fused cytokine protein is fused by either its amino-end or carboxyl-end with indicated region of immunoglobulin. Multifunctional fused protein cytokine-antibody shows an anticancer activity.
EFFECT: valuable medicinal properties of protein complexes.
13 cl, 40 dwg, 18 ex
FIELD: genetic engineering, proteins, medicine, pharmacy.
SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.
EFFECT: improved targeting method, valuable biological properties of fused protein.
10 cl, 5 dwg, 9 ex
FIELD: biotechnology, microbiology, medicine.
SUBSTANCE: method involves selection of signal sequence suitable for the effective expression of Leu-hirudine in E. coli cells by the polymerase chain reaction-screening method. Method involves construction of a protein as a precursor of hirudine based on the selected signal sequence of surface membrane protein from Serratia marcescens, oprF protein from Pseudomonas fluorescens or fumarate reductase from Shewanella putrifaciens by joining the Leu-hirudine amino acid sequence with C-end of selected signal sequence. Prepared precursor of Leu-hirudine is used in a method for preparing Leu-hirudine. Invention provides preparing Leu-hirudine by the direct secretion in E. coli cells with the high yield. Invention can be used in preparing the hirudine precursor.
EFFECT: improved preparing method.
4 cl, 1 dwg, 2 tbl, 12 ex
FIELD: biotechnology, medicine.
SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.
EFFECT: valuable properties of new cytokine.
18 cl, 5 tbl, 1 dwg, 55 ex
FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.
SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.
EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.
7 cl, 12 dwg, 4 tbl, 16 ex
FIELD: molecular biology, genetic engineering, polypeptides, medicine.
SUBSTANCE: in using the double-hybrid yeast system DNA sequences encoding polypeptides (55.1 and 55.3) have been found that elicit ability for binding with intracellular domain p-55 (p-55IC) of TNF-receptor. It has been established that these polypeptides represent fragments of amino acid sequences p-55IC, respectively, from 338 to 426 and from 277 to 426 residues. As result of insertion of DNA fragments with a sequence encoding polypeptide 55.1 or 55.3 into the structure of expressing vector and transformation suitable host-cells by this vector recombinant form of indicated polypeptides have been prepared. Using this invention provides the possibility for modulating the function of intact p-55 of TNF-receptor. Invention can be used in medicine in treatment of diseases associated with transfer of TNF-signal.
EFFECT: improved preparing method and valuable properties of polypeptide.
9 cl, 17 dwg, 3 tbl, 6 ex
FIELD: biochemistry, medicine.
SUBSTANCE: invention relates to two forms of peptide isolated from annelid worm (Arenicola marina) eliciting the broad spectrum of antibacterial effect. Indicated forms differ by a single amino acid residue at position 10 wherein at position 10 arenicin-1 has Val and arenicin-2 has Ile. Invention provides expanding assortment of antibacterial agents.
EFFECT: valuable medicinal properties of peptides.
1 tbl, 3 dwg, 4 ex
SUBSTANCE: invention proposes different compositions able to induce production of antibodies against Tat HIV-1 that can inhibit multiplication of HIV-1. Also, invention proposes a method for induction of antibodies raised against Tat HIV-1, in vitro method for assay of the presence of antibodies and their titer values, a method for reducing HIV-1 virus levels, sequence of synthetic nucleic acid and synthetic molecule. Proposed group of inventions can be used for inhibition of multiplication of HIV-1 in infected patients and for attenuation of HIV-1 multiplication after the primary infection in early infected persons.
EFFECT: valuable methods and compositions.
39 cl, 7 dwg, 9 tbl, 5 ex