MXPA97006418A - Ingap protein involved in neogenesis of the islote of pancr - Google Patents

Abstract

The cellophane (CW) envelope of the hamster pancreas induces the proliferation of epithelial duct cells followed by endocrine cell differentiation and islet neogenesis. When using the mRNA display technique, the cDNA clone expressed in the cellophane envelope but not in the control pancreas was identified. By using the cDNA as a probe, a cDNA library was purified and a gene was identified that had not been described before, which was named

Description

INGAP PROTEIN INVOLVED IN THE NEOGÉNESIS OF THE ISLOTE DEL PÁNCREAS BACKGROUND OF THE INVENTION The pancreatic islets of Langerhans are the only organ of insulin production in the body. However, they have a limited regeneration capacity. This limited regeneration capacity predisposes mammals to develop diabetes mellitus. Thus, in the technique of endocrinology there is a need for products that can stimulate the regeneration of the islets of Langerhans to prevent or alleviate the symptoms of diabetes mellitus. A model of pancreatic islet cell regeneration involves wrapping the golden Syrian hamster pancreas with cellophane (1). The envelope of the pancreas induces the formation of new endocrine cells that appear to originate from the duct epithelium (2-4). There is a need in the art to identify and isolate the factors that are responsible for the regeneration of the islet cell.

SUMMARY OF THE INVENTION An object of the invention is to provide a preparation of a mammalian protein or portions of E 478 polypeptide thereof involved in islet cell neogenesis. Another object of the invention is to provide a DNA molecule that encodes a mammalian protein involved in islet cell neogenesis. Another object of the invention is to provide a preparation of an INGAP protein (protein associated with islet neogenesis) of a mammal. Another objective of the invention is to provide nucleotide probes for detecting mammalian genes involved in islet cell neogenesis. An object of the invention is to provide a method for the isolation of INGAP genes from a mammal. Another objective of the invention is to provide an antibody preparation that is specifically immunoreactive with an INGAP protein. Another objective of the invention is to provide methods for producing INGAP proteins. An object of the invention is to provide methods for the treatment * of diabetic mammals. Another objective of the invention is to provide a method for the growth of pancreatic islet cells in culture. Another additional objective of the invention is to provide methods for increasing the life of cells of E'.78 pancreatic islet encapsulated in polycarbonate shells. An object of the invention is to provide methods for increasing the number of pancreatic islet cells of a mammal. An objective of the invention is to provide transgenic mammals. Another objective of the invention is to provide genetically engineered mammals. Another objective of the invention is to provide methods for identifying individual mammals at risk of diabetes. An object of the invention is to provide methods for detecting the INGAP protein in a mammalian sample. Another object of the invention is to provide a method for the treatment of isolated islet cells to prevent apoptosis. Another object of the invention is to provide methods for the treatment of mammals receiving islet cell transplants. An object of the invention is to provide a method for inducing differentiation of β-cell progenitors. An object of the invention is to provide a method for identifying cell progenitors /? E'.7H Another objective of the invention is to provide a method for the treatment of a mammal with pancreatic endocrine failure or problem. An object of the invention is to provide antisense constructs for regulating the expression of INGAP. Yet another objective of the invention is to provide a method for the treatment of nesidioblastosis. Yet another objective of the invention is to provide kits for detecting mammalian INGAP proteins. An object of the invention is to provide pharmaceutical compositions for the treatment of pancreatic insufficiency. These and other objects of the invention are provided by one or more of the embodiments described below. In a modality, a preparation of a mammalian INGAP protein is provided. The preparation is substantially free of other mammalian proteins. In another embodiment, a cDNA molecule encoding an INGAP protein is provided. In another embodiment of the invention, a preparation of mammalian INGAP protein is provided. The preparation is elaborated through the process of: inducing mammalian pancreatic cells to express INGAP protein, using cellophane wrap; Y purify INGAP protein from mammalian pancreatic cells induced.

In another embodiment of the invention, a nucleotide probe is provided. The probe comprises at least 20 contiguous nucleotides of the sequence shown in SEQ ID NO: 1. In another embodiment of the invention, an INGAP protein preparation of a mammal is provided. The preparation is purified substantially from other proteins of the mammal. The INGAP protein can be induced 'by wrapping the pancreas of the mammal with cellophane. In another embodiment of the invention, there is provided a method for isolating an INGAP gene from a mammal. The method comprises: hybridizing one or more oligonucleotides comprising at least 10 nucleotides contained in the sequence shown in SEQ ID NO: 1 with the DNA or cDNA of the mammal; identify DNA moles from the genomic DNA or cDNA that hybridize with one or more oligonucleotides. In still another embodiment of the invention, ee provides an isolated cDNA mole. The mole of CDNA is obtained by the process of: hybridizing one or more oligonucleotides comprising at least 10 contiguous nucleotides of the sequence shown in SEQ ID NO: 1 with the genomic DNA or cDNA of the mammal; identify DNA moles from the genomic DNA or cDNA that hybridize with one or more oligonucleotides.

In another embodiment of the invention, an antibody is provided. The antibody is specifically immunoreactive with a mammalian INGAP protein. In accordance with another embodiment of the invention, a method for producing a mammalian INGAP protein is provided. The method comprises the steps of: providing a host cell transformed with cDNA encoding a mammalian INGAP protein; culturing the host cell in a nutrient medium so that the INGAP protein is expressed; and harvesting the INGAP protein from the host cell or the nutrient medium. In accordance with another embodiment of the invention, there is provided a method for producing mammalian INGAP protein. The method comprises the step of: providing a host cell comprising a DNA mole obtained by the process of: hybridizing one or more oligonucleotides comprising at least 10 contiguous nucleotides of the sequence shown in SEQ ID NO: 1 with the Genomic DNA or cDNA of the mammal; identify DNA moles from the genomic DNA or cDNA that hybridize with one or more oligonucleotides; culturing the host cell in a nutrient medium so that the INGAP protein is expressed; and harvesting the mammalian INGAP protein from the host cell or the nutrient medium. In accordance with another embodiment of the invention there is provided a method for the treatment of diabetic mammals. The method comprises: administering to a diabetic mammal a therapeutically effective amount of an INGAP protein to stimulate the growth of islet cells. In accordance with another embodiment of the invention, a method is provided for the growth of pancreatic islet cells in culture. The method comprises: supplying an INGAP protein to a culture medium for the growth of pancreatic islet cells; and growing the islet cells in the culture medium comprising INGAP protein. In accordance with another embodiment in printing, a method is provided for increasing the life of pancreatic islet cells encapsulated in a polycarbonate shell. The method comprises: adding to encapsulated pancreatic islet cells an INGAP protein in an amount sufficient to increase the survival rate or survival time of pancreatic islet cells. In accordance with another embodiment of the invention, there is provided a method for increasing the number of pancreatic islet cells in a mammal. The method comprises: administering to the pancreas of a mammal a DNA mole that encodes an INGAP protein. In accordance with another embodiment of the invention, there is provided a method for increasing the number of pancreatic islet cells of a mammal. The method comprises: administering an INGAP protein to the pancreas of a mammal. In accordance with another embodiment of the invention, a transgenic mammal is provided. The mammal comprises an INGAP gene from a second mammal.

According to another embodiment of the invention, a non-human mammal is provided.The mammal has been genetically engineered to contain an insertion or mutation suppression of an INGAP gene of the mammal.In accordance with another embodiment of the invention, provides a method to identify individual mammals at risk of diabetes.The method comprises: identifying a mutation in an INGAP gene from a sample of an individual mammal, the mutation causes a structural abnormality in an INGAP protein encoded by the gene or causes a regulatory defect leading to a diminished or obliterated expression of the INGAP gene In accordance with another embodiment of the invention, a method for detecting INGAP protein in a mammalian sample is provided, the method comprising: contacting the sample with a antibody preparation that is specifically immunoreactive with a mammalian INGAP protein. In accordance with another embodiment of the invention, there is provided a method for the treatment of islet cells isolated from a mammal to prevent apoptosis of the cells. The method includes: 1' . IH contacting islet cells isolated from a mammal with a preparation of a mammalian INGAP protein, substantially purified from other mammalian proteins, in an amount sufficient to increase the survival rate of isolated islet cells . In accordance with another embodiment of the invention, there is provided a method for the treatment of a mammal receiving an islet cell transplant. The method comprises: administering a mammalian INGAP protein preparation to an islet cell transplant recipient mammal, wherein the step of administration is carried out before, during or after transplantation. In accordance with another embodiment of the invention, a method for inducing differentiation of β-cell progenitors is provided. The method comprises: contacting a culture of pancreatic duct cells comprising β-cell progenitors with a preparation of a mammalian INGAP protein substantially free of other mammalian proteins, to induce differentiation of beta-cell progenitors. In another embodiment of the invention, a method for the identification of β-cell progenitors is provided. The method comprises: contacting a population of pancreatic duct cells with a mammalian INGAP protein; and detect, from the population of cells, those to which the INGAP protein specifically binds. In accordance with another embodiment of the invention, a method is provided for the treatment of pancreatic endocrine failure or problem. The method comprises: contacting a preparation of pancreatic duct cells comprising β-cell progenitors isolated from a mammal suffering from pancreatic endocrine failure or problem with a preparation of a mammalian INGAP protein substantially free of other mammalian proteins, for induce the differentiation of ß cell progenitors; and transplant pancreatic product cells autologously into the mammal. In accordance with another embodiment of the invention, an antisense construct of a mammalian INGAP gene is provided. The construction comprises: a promoter, a terminator and a sequence M nucleotides consisting of a mammalian INGAP gene, the nucleotide sequence is between the promoter and the terminator, the nucleotide sequence is inverted with respect to the promoter, so that when the promoter is expressed, an mRNA complementary to the mRNA is produced INGAP of the native mammal. In accordance with another embodiment of the invention, a method for the treatment of nesidioblastosis is provided. The method comprises: administering to a mammal with nesidioblastosis an anti-sense construct, as described above, thereby inhibiting the overgrowth of the mammalian β-cells. In accordance with another embodiment of the invention, a kit is provided for detecting a mammalian INGAP protein in a mammalian sample. The kit comprises: an antibody preparation that is specifically immunoreactive with a mammalian INGAP protein; and a polypeptide comprising a sequence of at least 15 consecutive amino acids of a mammalian INGAP protein. In accordance with another embodiment of the invention, a pharmaceutical composition for the treatment of pancreatic insufficiency is provided. The composition comprises: a mammalian INGAP protein in a pharmaceutically acceptable diluent or carrier. In accordance with another embodiment of the invention, a pharmaceutical composition is provided. The composition comprises: a preparation of a polypeptide comprising a sequence of at least 15 consecutive amino acids of the mammalian INGAP protein or a pharmaceutically acceptable diluent or carrier. These and other embodiments of the invention provide the art with the means to stimulate and inhibit islet cell neogenesis. Also by means of this invention a means is provided for the diagnosis of minor conditions of diabetes mellitus.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. The nucleotide sequence of the hamster INGAP protein and the deduced sequence of the encoded immature protein. The non-coding sequences are in lowercase letters and the polyadenylation signal is underlined. Figure 2. Comparison of amino acid sequences of INGAP, PAP-I (PAP-I) (18) of rat, PAP / HIP F .i; - (PAP-H / HIP) (10, 11) of human, PAP-III (PAP-III) (9) of rat, PAP-II (PAP-II) (8) of rat, Reg (PSP / LitosLatina ( REG / LITH) (13, 15) and the invariable motif found by Drickamer in all members of type C lectins (Drickamer). (12) Six conserved cysteines are marked by asterisks and the 2 putative sites of N- GAP glycosylation is underlined and in bold type Figure 3. Northern blot assay of INGAP and expression of the amylase gene in pancreatic tissue from a control pancreas and a wrapped hamster pancreas. Total AR denatured by heat in 1.2% agarose, 0.6% formaldehyde / MOPS denaturing gel and transferred to a nylon membrane The membranes were hybridized with a hamster INGAP AD7c probe of 747 base pairs (FIG. cloned in our laboratory) (A), a rat amylase AD? c probe of 1000 base pairs (kindly provided by Chris? e Gard Dallas, Texas) (B) and with an 18S ribosomal synthetic oligonucleotide probe of 24 mer to monitor the integrity and loading (C) of AR ?.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Below is the identification of a gene, INGAP, which shows surprising homology with the f 4 gene family (7-11) of the protein associated with pancreatitis (PAP). The mentioned protein shares the carbohydrate recognition domain (CRD) of calcium-dependent type C lectins, as defined by Drickamer (12). The INGAP protein plays a role in the stimulation of islet neogenesis, in particular, in the regeneration of ß-cell from duct cells. The cDNA sequence of a mammalian INGAP is provided in SEQ ID NO: 1. The predicted amino acid sequence is shown in SEQ ID NO: 2. These sequences were determined from the nucleic acids isolated from the hamster but it is believed that other species of mammals will contain INGAP genes that are very similar. The human INGAP cDNA shares the sequence from 23 to 268 and 389 to 609 in SEQ ID NO: 1 with a gap or gap of 159 base pairs in the middle of the sequence. The aforementioned amino acid sequence of the human INGAP protein ranges from 1 to 83 and from 124 to 174 in SEQ ID NO: 2 with 53 amino acids in the middle of the sequence. It is expected that the homologous genes contain at least about 70% identity. It is expected that the closest species have at least approximately 75%, 89% or even 85% identity. In contrast, other members of the family of type C lertins dependent on calcium contain a maximum of 60% identity with INGAP. The DNA sequence provided herein can be used to form vectors that will replicate the gene in a host cell and can also express INGAP proteins. DNA sequences encoding the same amino acid sequence as shown in SEQ ID NO: 2 could also be used, without deviating from what was contemplated in the invention. DNA sequences encoding other mammalian INGAPs are also contemplated by the invention. Suitable vectors, both for prokaryotic cells and for eukaryotic cells, are known in the art. Some vectors are specifically designed to effect the expression of DNA segments inserted downstream of a transcription and translation control site. One of these vectors for expression in eukaryotic cells employs EBNA His, a plasmid that is commercially purchased from In Vitrogen Corp. The loaded vector produces a fusion protein comprising a portion of a histidine biosynthetic enzyme and INGAP. Another vector that is suitable for use in prokaryotic cells is pADNc3. The selection of a vector for a particular purpose can be done using knowledge of the properties and particularities of vectors, such as for example useful expression control sequences. The vectors can be used to transform or to transfect host cells, either stably or transiently. Transformation and transfection methods are known in the art and can be used in accordance with the suitability of a particular host cell. The host cells can be selected in accordance with the purpose of the transfection. A suitable prokaryotic host is E. Coli DH5a. A suitable eukaryotic host is cos7, a kidney cell line of the African Green Monkey. For certain purposes the appropriate glycosylation of the INGAP may be desired, in which case, a suitable host cell recognizing the glycosylation signal of INGAP should be used. Lae probes comprising at least 10, 15, 20 or 30 of contiguous sequence according to SEQ ID NO: 1 can be used as INGAP genes in particular individuals or in members of other species. In the art, inappropriate conditions for DNA hybridizations of the species or different species are known as high restriction and low restriction, respectively. These can be used in a variety of formats in accordance with the intended use. For example, Southern blot, Northern blot, and colony hybridization assays may be used as they are known in the art. The probes are usually oligomers of DNA or RNA of at least 10, 15, 20 or 30 nucleotides. The probe can be labeled with any detectable entity known in the art, including radioactive labels, fluorescent labels, enzymes, etc. The probes can also be derived from other mammalian INGAP gene sequences. The INGAP genes can be isolated from other mammals using the nucleotide sequence information provided therein (more laboriously, they can be isolated using the same method described in detail below for the isolation of the hamster INGAP gene). Oligonucleotides comprising at least 10 contiguous nucleotides of the presented nucleotide sequence of the INGAP are hybridized with the genomic DNA or cDNA of the mammal. The DNA can conveniently be in the form of a clone bank. The oligonucleotides can be labeled with any convenient marker, such as for example a radioactive label or an enzyme or flurorescence label. The DNA molecules that hybridize to the probe are isolated. Whole genes can be constructed by isolating the overlapping DNA segments, for example using the first asylated DNA as a probe with the contiguous DNA in the gene bank or by preparing the mammalian DNA. Confirmation of the identity of the isolated DNA can be effected by observing the expression pattern of the gene in the pancreas when it is subjected to the cellophane wrap, for example. Similarly, the biological effect of the product encoded on the pancreatic duct cells will also serve to identify the gene as an INGAP gene. If two oligonucleotides are hybridized with the genomic DNA or cDNA of the mammal, then primers can be used for DNA synthesis, for example, using the polymerase chain reaction or the ligase chain reaction. The construction of a full-length gene and confirmation of the identity of the isolated gene can be performed as described above. The INGAP protein can be isolated according to the invention by inducing mammalian pancreatic cells to express INGAP protein by means of cellophane wrapping. This technique is described in detail in reference no. 1 which is expressly incorporated herein. The INGAP protein produced in this way can be purified from other mammalian proteins by means of immunoaffinity techniques, for example, or other techniques known in the field of protein purification. An antibody specific for a mammalian INGAP is produced using all or fragments of the amino acid sequence of an INGAP protein, such as for example the one shown in SEQ ID NO: 2 as immunogens. Immunogens can be used to identify and purify immunoreactive antibodies. Monoclonal or polyclonal antibodies can be prepared as is well known in the art. The antibodies can be conjugated to other entities, such as for example detectable labels or solid support materials. These antibodies can be used to purify proteins isolated from mammalian pancreatic cells or from recombinant cells. Hybridomas that secrete specific antibodies to an INGAP protein are also within the contemplation of the invention. The host cells as described above can be used to produce a mammalian INGAP protein. The host cells comprise a DNA molecule that encodes a mammalian INGAP protein. The DNA can be made in accordance with SEQ ID NO: l or isolated from other mammals in accordance with the methods described above. The host cells can be cultured in a nutrient medium under conditions where the INGAP protein is expressed. The INGAP protein can be isolated from host cells or from the nutrient medium, if the INGAP protein is secreted from host cells. It has now been found that INGAP and its fragments are able to induce and stimulate the growth of islet cells. In addition, they are capable of inducing the differentiation of pancreatic duct cells and allowing cells to avoid the apoptotic path. Thus, many therapeutic modalities are currently possible using the INGAP protein, fragments thereof and nucleotide sequences that encode it. Amounts of INGAP that are therapeutically effective are delivered to the patient's pancreas, to isolated islet cells and to encapsulate the pancreatic islet cells, such as, for example, in a polycarbonate shell. Adequate amounts of INGAP for therapeutic purposes range from 1-15 μg / kg body weight or in the range of 1-10,000 μg / ml. The optimization of these doses can be determined by routine tests. The methods of administration of INGAP to mammals can be any of those known in the art, including subcutaneous, through the portal vein, by local perfusion, etc. The conditions that can be treated according to the invention by supplying the INGAP include diabetes mellitus, both insulin dependent and non-insulin dependent, pancreatic insufficiency, failure or pancreatic problems, etc. Inhibition of INGAP expression can use nesidioblastoeie. In accordance with the present invention, it has now been found that a small portion of INGAP is sufficient to confer biological activity. A fragment of 20 amino acids of the sequence of SEQ ID NO: 2, amino acid # 103 to # 122 is sufficient to stimulate pancreatic duct cells to grow and proliferate. The effect has been observed in a rat tumor duct cell line, in a hamster duct cell line, in a hamster insulinoma cell line and in a rat insulinoma cell line. It is very likely that analogous portions of the other mammalian INGAP proteins have the same activity.

This portion of the protein is not similar to other members of the protein family associated with pancreatitis (PAP). It contains a glycosylation site and is probably also a primary antigenic site of the protein. This fragment has been used to immunize mice to generate monoclonal antibodies. The physiological site of expression of INGAP has been determined. INGAP is expressed in acinar tissue, in the exocrine portion of the pancreas. It is not expressed in duct or islet cells, that is, the paracrine portion of the pancreas. The expression occurs within 24-48 hours of induction by cellophane wrap. The transgenic animals according to the present invention are mammals that carry an INGAP gene from a different mammal. The transgene can be expressed at a higher level than the endogenous INGAP genes through the judicious selection of the regulatory regions of the tracification. The methods for preparing transgenic animals are well known in the art and any of these methods can be used. Animals that have been genetically engineered to carry insertions, deletions and other mutations that alter the structure of the INGAP protein or the regulation of INGAP expression are also contemplated by this invention. The techniques for effecting these mutations are known in the field of the invention. Diagnostic assays are also contemplated within the scope of the present invention. Mutations in INGAP can be determined in samples, such as blood, amniotic fluid, chorionic villus, blasts, and pancreatic cells. These mutations identify individuals who are at risk for diabetes. Mutations can be identified by comparing the nucleotide sequence with a wild-type sequence in an INGAP gene. This can be done E 1 by any technique known in the art, including the comparison of polymorphs of the length of restriction fiaginet, the comparison of polymerase chain reaction products, nuclease protection assays, etc. Alternatively, the altered proteins can be identified, for example, immunologically or biologically. The present invention also contemplates the use of INGAP antisense constructs for the treatment of nesidioblastosis, a condition characterized by the overgrowth of β-cells. The antisense construct is administered to a mammal that has nesidioblastosis, thereby inhibiting the overgrowth of the β-cells. An antisense construct typically comprises a promoter, a terminator and a nucleotide sequence consisting of the mammalian INGAP gene. The INGAP sequence is between the promoter and the terminator and is inverted with respect to the promoter as it is naturally expressed. With the expression of the promoter, a mRNA complementary to the native mammalian INGAP is produced. Immunological methods for the INGAP assay in a sample from a mammal are useful, for example, to monitor the therapeutic administration of INGAP. Normally, an antibody specific for INGAP will be put in contact with the sample and the binding between the antibody and any INGAP in the sample will be detected. This can be by a competitive binding assay in which the incubation mixture is seeded with a known amount of a standard INGAP preparation, which can conveniently be detectably labeled. Alternatively, an INGAP polypeptide fragment can be used as a competitor. In a particular assay format, the antibodies bind to a solid phase or support, such as for example a bead, a polymer matrix or a microtiter plate. In accordance with the present invention, pancreatic duct cells of a mammal with pancreatic endocrine failure or problem can be removed from the body and treated in vi tro. The duct cells normally comprise β-cell progenitors. In this way, treatment with a preparation of a mammalian INGAP protein will induce the differentiation of ß cell progenitors. The conduit cells are contacted with a preparation of a mammalian INGAP protein substantially free of other mammalian proteins. The treated cells can then be used as an autologous transplant in the mammal from which they were derived. This autologous treatment minimizes adverse host reactions against the graft reactions involved in the transplants.

The INGAP protein can also be used to identify those cells that poIL nír opl 011 > . paiu rL INGAP. These cells are likely to be β-cell progenitors, which are sensitive to the biological effects of INGAP. The INGAP protein can be labeled in detectable form, such as with a radioactive label or a fluorescent label, and then contacted with a population of pancreatic duct cells. The cells that bind to the labeled protein will be identified as those that carry receptors for INGAP and, thus, are ß cell progenitors. The fragments of INGAP can also be used for this purpose, like the immobilized INGAP that can be used to separate cells from a mixed population of cells on a solid support. INGAP can be immobilized in solid phase or support by adsorption to a surface, by means of an antibody or by conjugation. Any other means as known in the art can also be used. By means of the present invention, kits for the detection of a mammalian INGAP protein in a sample are provided. This may be useful, inter alia, to monitor the metabolism of INGAP during therapy, which involves the administration of INGAP to a mammal. The kit will normally contain an antibody preparation that is specifically in unreactive with a mammalian INGAP protein. The antibodies can be polyclonal or monoclonal. If they are polyclonal they can be purified by affinity to make them onospecific. The kit will normally also contain a polypeptide having at least 15 consecutive amino acids of a mammalian INGAP protein. The polypeptide is used to compete with the INGAP protein in a sample to bind to the antibody. Desirably, the polypeptide will be labeled in detectable form. The polypeptide will contain the INGAP information to which the antibody binds. Thus, if the antibody is monoclonal, the polypeptide will compete successfully with the INGAP because it contains the antibody epitope. It may also be desirable for the antibodies to bind to a solid or support, such as beads, sticks, polymer plaques, etc. Pharmaceutical compositions containing a mammalian INGAP protein can be used for the treatment of pancreatic insufficiency. . The composition may alternatively contain a polypeptide that contains a sequence of at least 15 consecutive amino acids of a mammalian INGAP protein. The polypeptide will contain a portion of INGAP that is biologically active in the absence of the other portions of the protein. The polypeptide may be part of a larger protein, such as a genetic fusion with a second protein or polypeptide. Alternatively, the polypeptide can be conjugated to a second protein, for example, by means of a cross-linking agent. Suitable portions of INGAP proteins can be determined by homology to amino acids # 103 to # 122 of SEQ ID NO: 2 or by the ability to test polypeptides to stimulate pancreatic duct cells to grow and proliferate. As is known in the art, it is often the case that a relatively small number of amino acids can be removed from either end of a protein without deconstructing the activity. Thus, within the scope of the invention it is contemplated that up to about 10% of the protein can be suppressed and still essentially provide all the INGAP functions. These proteins have at least about 130 amino acids, in the case of hamster INGAP. The pharmaceutical composition will contain a pharmaceutically acceptable diluent or carrier. A liquid formulation is generally preferred. INGAP can be formulated at different concentrations or using different formulations. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers or regulators, albumin, surfactant or acjont 0-5 dc volume. Carbohydrates preferably include sugar or sugar alcohols, such as, for example, mono-, di-, or polysaccharides or water-soluble glycans. The saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xyloea, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose, or mixtures thereof. Sucrose is the most preferred. The sugar alcohol is defined as a hydrocarbon of C to C ^ having an -OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. The one he prefers the most is mannitol. These sugars or sugar alcohols mentioned above can be used individually or in combination. There is no fixed limit to the amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. Preferably, the concentration of sugar or sugar alcohol is between 1.0% w / v and 7.0% w / v, more preferably, between 2.0 and 6.0% w / v, preferably, the amino acids include carnivorous (L) forms of carnitine , arginine and betaine; however, other amino acids may be added. Preferred polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000 or polyethylene glycol (PEG). With an average molecular weight between 3,000 and 5,000. It is also preferred to use a buffer or regulator in the composition to minimize pH changes of the solution prior to lyophilization and after reconstitution, if used. Almost any buffer or physiological regulator can be used, but citrate, phosphate, succinate and glutamate regulators or mixtures thereof are preferred. Preferably, the concentration ranges from 0.01 to 0.3 molar. Surfactants can also be added to the formulation. In addition, the INGAP or the polypeptide portion thereof can be chemically modified by covalent conjugation with a polymer to increase its half-life in circulation, for example. Preferred polymers and methods for binding them to peptides are shown in U.S. Patent Nos. 4,766,106, 4,179,337, 4,495, 285 and 4,609,546. The preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: R (0CH ^ -CH) m0 -R where R can be sei. hydrogen or a protecting group, for example an alkyl or alkanol group. Preferably, the protecting group has between 1 and 8 I 1 carbons, more preferably it is methyl. The symbol n is a positive integer, pieleonontum ont-r 1 and 1,000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1,000 and 40,000, more preferably between 2,000 and 20,000 and with the greatest preference between 3,000 and 12,000. Preferably, the PEG has at least one hydroxy group, more preferably it is a hydroxy terminal group. It is this hydroxy group which is preferably activated to react with a free amino group in the inhibitor. After the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility. Methods for lyophilization of liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition can be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to the subjects using those methods that are known to those skilled in the art. It is not intended that the following examples limit the scope of the invention but merely exemplify what was disclosed above.

EXAMPLES Example 1 This example describes the cloning and isolation of a cDNA encoding a novel pancreatic protein regulated experimentally. We propose the hypothesis that a single factor (factors) locally produced is responsible for the regeneration of the islet cell. Using the newly developed mRNA differential display or display technique (5,6) to compare genes expressed differentially in cellophane wrap (CW) versus control pancreas (CP) allows us to identify a cDNA clone (RD19-2) that was expressed only in cellophane envelope pancreas. A cDNA library was constructed from mRNA isolated from hamster pancreas with cellophane wrap using the primed oligo d (T) synthesis and ligated into the pcDNA3 vector (Invitrogen). The number of primary recombinants of the cDNA library was 1.2 X 106 with an average size of 1.1 kb. The cDNA library was purified to obtain the clones of interest, using techniques for the plating of high density colonies. The colonies were raised on nylon membranes (Schleicher &Schuell) and subsequently digested with proteinase K (50 (g / ml) .The treated membranes were baked at 80 ° C for 1 hour and hybridized at 50 ° C for 16 hours. -18 hours with 1-5 x 10ß cpm / ml RD19-2 labeling -adioactdvo [(3-P] -dCTP (Dupont -New England Nuclear) .Colonies with a positive hybridization signal were isolated, their Size was compared to Northern mRNA transcription and sequenced to confirm identity with the RD19-2 sequence.

Example 2 This example compares the sequence of INGAP with other proteins with which it shares homology. The nucleotide sequence of hamster INGAP was cloned with the longest cDNA insert. As shown in Figure 1, the hamster cDNA comprises 747 nucleotides (nt), excluding the poly (A) tail and contains a main open reading frame encoding a protein of 175 amino acids. The open reading frame is followed by a 3 '-not translated region of 206 nt. A typical polyadenylation signal is present at 11 nt upstream of the poly (A) tail. The predicted INGAP protein shows structural homology with both the PAP / HIP gene family that is associated with pancreatitis or with liver adenocarcinoma (7-11) and with the Reg / PSP / litostatin gene family (13, 15) that is ee it has been shown to stimulate the growth of the pancreatic beta cell (14) and may play a role in the regeneration of pancreatic islet. Comparison of the sequence of nucJ cutids and their reduced amino acids between hamster INGAP and rat PAP-I shows a high degree of homology in the coding region (60 and 58% in the nucleotide and amino acid sequence)., respectively). The predicted amino acid sequence of hamster INGAP reveals 45% identity with PAP II and 50% with PAP III, both of which have been associated with acute pancreatitis, and 54% with HIP that was found in hepatocellular carcinoma. INGAP also exhibits 40% identity with the Reg protein / PSP / rat lithostatin (Figure 2). The Reg is thought to be identical to the pancreatic stone protein (PSP) (15,16) or pancreatic cord protein (PTP) (17). The N term of the predicted sequence of the I? GAP protein is very hydrophobic which makes it a good candidate to be the signal peptide that would allow the protein to be secreted. Similar to PAP / HIP but different from Reg / PSP / litostatin proteins, a potential N-glycosylation site is located at position 135 of the I? GAP sequence. Unique I? GAP is another potential site for N-glycosylation located at position 115. The I? GAP also shows a high degree of homology (12/18) (Figure 2) with a consensus motif in the members of the calcium-dependent animal letty (type C), as determined by Drickamer, including four perfectly conserved cysteinee that form two linkages of i sul was or (1.1). Two additional cysteines found in the amino terminus of the INGAP (Figure 2) are also present in Reg / PSP and PAP / HIP. However, it is not clear what the biological meaning would be.

Example 3. This example demonstrates the temporal expression pattern of the INGAP with cellophane wrap. In order to determine the temporal expression of the INGAP gene, the total RNA extracted from the CP and CW pancreas was probed with the hamster INGAP cDNA clone in the Northern blot analysis. A single strong transcript of 900 base pairs was detected (Figure 3) 1 and 2 days after the cellophane wrap that disappeared from 6 to 42 days and was absent from the CP. INGAP mRNA is associated with pancreatic islet neogenesis with induced CW, since it is present only after CW. It is unlikely that the increase in expression in the INGAP is associated with acute pancreatitis as is the case with the PAP gene family. During the acute phase of pancreatitis, the concentrations of most pancreatic enzymes that encode mRNA including amylase are significantly decreased (16, 18). In contrast, in the CW model of islet neogenesis in which a high or high expiration of INGAP has been detected, the expression of the amylase gene ee increased simultaneously above normal (Figure 3) instead of decreasing, suggesting that the INGAP expression is not associated with pancreatitis but rather with islet neogenesis. The cause of the increase in the expression of the amylase gene 1 and 2 days after the CW is not clear yet and further studies need to be carried out to clarify this issue. Although it is unlikely that the increase is associated with exocrine cell regeneration that occurs at a later time after the CW (19). In this way, the INGAP protein plays a role in the stimulation of islet neogenesis, in particular, in the regeneration of beta cell duct cells.

Example 4 This example describes the cloning and partial sequence of an INGAP protein encoding human cDNA. Human polyA + RNA was isolated from a normal human pancreas using a polyA extraction kit purchased from Qiagen. Subsequently, they were used 500 ng of polyA RNA as a template for reverse transcription and the chain reaction of the 1 polymerase (RT-PCR). The experimental conditions were adjusted in accordance with the instructions of the Perkin Elmer RT-PCR kit. As a primer in the reverse transcription Oligo d (T) was used. As primers specific for the polymerase chain reaction, the primers corresponding to nucleotides 4 to 23 and 6120 to 629 were used in SEQ ID NO: 1. A PCR fragment of 626 base pairs was cloned using a TA cloning kit. Invitrogen. The partial sequence of the human clone comprises 466 base pairs with a gap or gap of 120 base pairs in the middle of the sequence. The human INGAP cDNA is 100% identical to the hamster INGAP cDNA sequence from nucleotide 4 to 268 and from nucleotide 289 to 629 in SEQ ID NO: 1. The sequence of the 120 base pairs in half remains unidentified.

EXAMPLE 5 This example demonstrates that synthetic peptides from INGAP play a role in the stimulation of islet neogenesis and that at least one epitope encoded by the 120 base pair segment, not yet sequenced, of the INGAP is shared with the INGAP of Hamster A synthetic peptide corresponding to amino acids 140-118 in SEQ ID NO: 2 of the deduced hamster INGAP protein was used as an immunogen to raise polyclonal antibodies in a rabbit. The antisue, ro was subsequently used in the immunohistochemical assays using the avidin-biotin complex (ABC) method. Cells in the peri-islet region in humans with neoislote formation stained positively for INGAP demonstrating that the human and hamster INGAP share a common epitope between amino acids 104 to 118 in SEQ ID NO: 2. The ability was tested of the same synthetic peptide to stimulate the incorporation of? -thymidine in rat pancreatic tumor duct (ARIP) cells and in inoma hamster insul tumor (HIT) cells. LOμCi of H-thymidine was added at a concentration of 80.4 Ci / mmol to approximately 10'cultured cells in Ham's F-12K medium. After 24 hours, the cells were harvested and solubilized. The differential precipitation of the nucleic acid with trichloroacetic acid (TCA) was carried out according to the procedure modified by Tosenberg et al. and the proportion of incorporated 3H-thymidine was calculated. The addition of the synthetic peptide to the ARIP culture resulted in an increase of 2.4 in the incorporation of H-thymidine compared to the absence of the synthetic peptide in the culture. The synthetic peptide did not have E'4 effect on the HIT of the control cell line. This result strongly suggests that INGAP plays a role in the stimulation of islet neogenesis. r I REFERENCES 1. Rosenberg, L., Brown, R.A. and Duguid, W.P. (1982). Surg. Forum 33, 227-230, 2. Rosenberg, L., Brown, R.A. and Duguid, W.P. (1983). J. Surg. Res. 35, 63-72. 3. Rosenberg, L., Duguid, W.P. and Vinik, A. I. (1987). Dig. Dis. Sci. 37, 1185. 4. Cias, D., Rosenberg, L., and Duguid, W.P., (1989). Pancreas 4, 613 (Extract). 5. Liang, P. and Pardee, B.A. (1992). Science 257, 967-971. 6. Liang, P., Averboukh, L. and Pardee, B.A. (1993). Nucleic Acid Res. 21, 3269-3275. 7. Iovanna, J. Orelle, B., Keim, V. and Dagorn, J.C. (1991). J. Biol. Chem. 266, 24664-24669. 8. Frigerio, J.M., Dusetti, N., Keim, V., Dagorn, J.C. and Iovanna, J. (1993). Biochemistry 32, 9236-9241. 9. Frigerio, J.M., Dusetti, N., Garrido, P., Dagorn, J.C. and Iovanna, J. (1993). Biochim. Biophy. Acta 1216, 329-331. 10. Orelle, B., Keim, V., Maeciotra, L., Dagorn, J.C. and Iovanna, J. (1992). J. Clin. Invest. 90, 2284-2291. 11. Laseerre, C, Christa, L., Simon, M.T., Vernier, P. and Brechot, C. (1992). Cancer Res. 52, 5089-5095. 12. Drickamer, K. (1988). J. Biol. Chem. 263, 9557-9560. 13. Terazono, K., Yamamoto, H. Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y. and Oka oto, H. (1988). J. Biol. Chem. 263, 2111-2114. 14. Watanabe, T., Yutaka, Y., Yonekura, H., Suzuki, Y., Miyashita, H., Sugiyama, k., Morizumi S., Unno, M., Tanaka, 0., Kondo, H., Bone, A.J., Takasawa, S. and Okamoto, H. (1994). Proc. Natl. Acad. Sci. USA 91, 3589-3592. 15. Rouquier, S., Giorgi, D., Iovanna, J. and Dagorn, J.C. (1989). Biochem. J. 264, 621-624. 16. Rouquier, S., Verdier, J., Iovanna, J., Dagorn, J.C. and Giorgi, D. (1991) J, Biol. Chem. 266, 786-791. 17. Gross, J., Carlson, R.I. Brauer, A.W., Margolies, M.N., Warshaw, A.L. and Wande, J.R. (1985). J. Clin. Invest. 76, 2115-2126. 18. Iovanna, J., Keim, V., Michael, R. and Dagornt J.C. (1991) Am. J. Physiol. 261, G485-G489. 19. Rosenberg, L. and Vinik, A. I. (1989). J. Lab. Clin. Med, 114, 75-83.

! I LISTINGS OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Eastern Virginia Medical School delf the Medical College of Hampton Roads McGill University (ii) TITLE OF THE INVENTION: PROTEIN INGAP THAT INTERVENES IN THE NEOGENYSIS OF THE ISLAND OF THE PANCREAS (iii) NUMBER OF SEQUENCES: 7 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Banner & Allegretti, Ltd. (B) STREET: 1001 G Street, N.W. (C) CITY: Washington (D) STATE: D.C. (E) COUNTRY: E.U.A. (F) POSTAL CODE: 20001-4597 (v) FORM FOR COMPUTER READING: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS ( D) SOFTWARE: Patentln Reléase # 1.0, Version # 1.25 (vi) DATA OF THIS APPLICATION (A) NUMBER OF APPLICATION: (B) DATE OF SUBMISSION: 12 -FEBRERO-1996 (C) CLASSIFICATION: (viii) INFORMATION OF THE EMPLOYEE / AGENT (A) NAME: Kagan, Sarah A. (B) REGISTRATION NUMBER: 32,141 (C) REFERENCE / RECORD: 00570.54144 (ix) INFORMATION FOR TELECOMMUNICATION (A) PHONE: 202-508-9100 EM > - (B) TELEFAX: 202-508-9299 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 747 base pairs \ (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Cricetulus (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 20.541 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: CTGCAACAC? GGTACCATG ATC CTT CCC ATC ACC CTC TGT AOO ATG TCT TCG 5 Het Leu Fro Het Thr Lau Cyu Ara Mßt Sor Trp 1 5 10 ATG CTC CTT TCC TGC CTG ATO TTC CTT TCT TCG GTG C ?? GGT G? A GAA 100 Mot Leu Leu Ser Cye Leu Met Pho Lou Ser Trp Val slu sly Glu slu 15 20 25 TCT CAA AAG AAA CTG CCT TCT TCA CCT ATA ACC TCT CCT C? A GGC TCT 140 Be aln Lys Lye Leu Fro Ser Sßr Arg lie Thr Cyp Pro Qln Gly be 30 35 40 CTA GCC TAT &GG TCC TAT TGC TAT TCA CTG ATT TTC? T? CCA CAG ACC 196 Val Wing Tyr Gly Ser Tyr Cye Tyr Sßr Leu He Lou lie Pro Gln T r 45 SO 55 TCO TCT AAT GCA GAA CTA TCC TGC CAO ATG C? T TTC TC? DC? CAC CTG 244 Trp Ser Aon? La Clu Leu Sor Cyo Cln Mot Hio Fho Sor Gly Hio Lou 60 65 70 75 IM > > OCA TTT CTT CTC? CT? CT GGT G ?? ? TT? CC TTC CTG TCC TCC CTT GTG 292? The Pho Leu Leu Ser Thr Gly Glu He Tht The Val Ser Sor Leu Val BO 65 90 AAG AAC AGT TTG ACG CCC TAC C? C T? C? TC TGG? TT GO? CTC C? TC? T 340 Lyß Apn Sister Lou Thr? Tyr Oln Tyr lio Trp He Gly Leu Hiu? Op 95 100 105 CCC TCA CAT OQT ACA CTA CCC AAC GGA AGT CCA TCC? AG TCG ACC? CT 300 Pro Ser Hio Gly Thr Leu Pro Aon sly Eer aly Trp Ly? Trp Sor Ser 110 115 120 TCC A? T OTO CTO ACC TTC TAT AAC TOO GAO? GG A? C CCC TCT? TT CCT 436 Ser Aßn Val Leu Thr Ph? Tyr Aan Trp Glu? Rg? On Pro Ser He? La 125 130 135 GCT C? C CGT GGT TAT TOT GCA GTT TTG TCT C? G? A? TC? GGT TTT CAG 484 Wing App Arg Gly Tyr CyB Wing Val Leu Ser Cln Lyo Sor Gly Pha Cln 140 145 150 155 AAG TGG AGA CAT T T AAT TCT C ?? ?? T 0? 0 CTT CCC TAT? TC TOC ??? 53? Lyß Trp Arg A »p Phe Aan Cya Clu? On Qlu Leu Pro Tyr He Cy? Lyo 160 165 170 TTC A? G GTC TAOOGCAOTT CTAATTTCAA CAOCTTO? A? ? T? TT? TG ?? 581 Ph? LyB Val GCTCACATGG AC? G A? GC? ACT? TOAGG ATTCACTCAC C? ACAGCA? C CTCTGCCT? C 641 ACACCCACAC CAATTCCCTT ATATC? TCTC TGCTGTTTTT CTATC? GT? T? TTCTGTCOT 701 aaCTQTAACC T? AACrGCTCA GAG ?? C ???? ? T? A? TOTC? TCA? C 747 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 174 amino acid residues (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: FM Mßt Lou Pro Met Thr Leu Cya Arg Net Eer Trp Met Lou Leu Ger Cyß 1 5 10 15 Lßu M? T Ph? Lou Ser Trp Val Olu Gly Glu Glu Ser Gln LyB Lya Lou 20 25 30 Pro Ser Ser Arg Ile Thr Cya Pro Gln Gly ser Val Ala Tyr Cly eer 35 40 45 Tyr Cya Tyr Sor Leu He L? U He Pro Cln Thr Trp Ser ? ßn? the Glu 50 55 60 Leu Ser Cyu Gln M? b Hiß Phe Ser Gly Hip Leu Ala Phe Leu Leu Ser 65 70 75 BO Thr Gly Glu lie Thr Phe Val Ser Ser Leu Val Lyo? On Ser Leu Thr B5 90 95 Wing Tyr Oln Tyr He Trp lie Gly Leu HIB Aßp Pro Ser Hiß Gly Thr 100 105 110 Leu Pro A? N Qly Ser Cly Trp Lyp Trp Ser Ser Aßn Val Leu Thr 115 120 125 Ph? Tyr? An Trp Glu Arg Aon Pro Ser He? La Ala? Op? Rg Gly Tyr 130 135 140 Cyß Ala Val LTU Sßr aln LyD Ser sly Phe Cln Lyß Trp? Rg ?? p Pho 14B 150 155 160 Aßn Cyß Glu Aan Clu Leu Pro Tyr lie Cyß Lyß Phe Ly? Val 165 170 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 175 amino acid residues (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 EM7H Met Lou Hio? Rg Leu? The Ph? Pro Val Met Sor Trp M..L Leu Leu Ser 1 5 10 15 Cyo L? U M? T Leu Leu Ser Cln Val Oln Gly Glu? Op Sc-r Ly »Lyo 20 25 30 lio Pro Sar Ala Arg He Ser Cya Pro Ly? sly Ser Gln? ln Tyr Gly 35 40 45 fler 'Tyr Cys Tyr Ala Leu Phe Gln Il? Pro Gln Thr Trp Phe? Ep? 50 55 60 Glu Leu? CyB Gln Lya Arg Pro Glu Gly Hio Leu Val Ser Val Leu 65 70 75 B0 Even Val Ala Glu Ala Sar P..T Lou? La Sar Met Val Ly-- Ann Thr sly 85 90 95 Aan Ser Tyr Gln Tyr He Trp lio Cly Leu Hi? Aßp Pro Thr LPU sly 100 105 110 Gly Glu Pro Aan Gly Gly Gly Trp Glu Trp Ser? On have Anp He Met 115 120 125 Aan Tyr Val Aßn Trp Glu Ar? Aon Pro be Thr Ala Leu? Pp Aro sly 130 135 140 Ph? Cyß Gly Ser Leu Ser Arg Ser Ssr Gly Phe Leu? Rs Trp? Rs? Op 145 150 15S 160 Thr Thr Cyß Glu Val Lya Leu Pro Tyr Val Cya Lyn pho Thr Gly 165 170 175 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 175 amino acid residues (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: And IH Met Leu Pro Pro M? T Ala Leu Pro s? R Val Ser Trp Hot Lau LHU Sister 1 5 10 15 Cys Leu Met Leu Leu Ser aln Val Gln Cly Olu Olu Pro Cln? R ?; Glu 20 25 30 Leu Pro Sister Wing Arg He Arg Cyß Pro Ly? Gly Ser Lyo Wing Tyr Gly 35 40 45 Ser HlD Cyo Tyr Ala Leu Phe Leu Ser Pro Lyo Ser Trp Thr Aep? La 50 55 50 Aßp Leu Ala Cy Gl Gln Lyo Arg Pro Ser Cly? Sn Leu Val Ser Val Leu 65 70 75 60 be Cly Wing Glu sly Being Phß Val Being eer Leu Val Lye Being He Gly GS 90 95 Aan Being Tyr Being Tyr Val Trp He Cly Leu Hip? pp Pro Thr Gln sly 100 105 HO Thr Clu Pro Aßn Cly Clu Cly Trp slu Trp Ser Sister Ser? Op Val Mot 115 120 Aßn Tyr Pho Wing Trp Glu? Rg AB? Pro Ber Thr He Sor Ser Fro Gly 130 135 140 Hiß Cyß Wing Ser Leu Ser Arg Ser Thr? Phe Lou Arg Trp Lyn? Sp 145 150 155 160 Tyr Aon Cyo? On Val? Xg Leu Pro Tyr Val Cy? Lyo Phe Thr Aop 165 170? 75 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 174 amino acid residues (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Mat e Pro Arg Val? The Leu Thr Thr Met Being Trp Met Lou Lou Being 1 5 10 15 Being Leu Met Leu Leu Being sln Val Cln Cly Clu? Tp Wing Lyp Clu? Op 20 25 30 Val, Pro Thr sßr Arg Ilß Ser Cyß Pro Lyß sly Ser? Rg? The Tyr Gly 35 40 45 Ser Tyr Cyß Tyr? The Leu Fhe Sor Val Sc.r Lys Ser Trp Phe? Pp? L 50 55 60 App Leu Ala Cyß Gln Lya? Rg Fro eer sly Hio Leu Val Ser val Lou 65 70 75 80 Ser Cly Ser Clu Wing Ser Pho Pho Sister Sai L? U lio LyD Ger Ser Gly 85 90 95 Aßn Ser Oly Gln Aon Val Trp lio sly Lou Hio? Op Pro T r Lou sly 100 105 1.10 Oln Glu Pro Aßn Arg Gly Gly Trp Clu Trp B? R A? N Ala? Op Val M? T 115 120 125 Aßn Tyr Phe Aon Trp Glu Thr Asn Pro Ser Sor Val Ser Gly Ser Iliß 130 135 J 0 Cya Gly Thr Leu Thr Arg Ala Ser Gly Pha Leu? Rg Trp? Rs slu Asm 145 150 155 JGo Aan Cyß Ilß Ser Glu Leu Pro Tyr Val Cyo LyD Pho Lyu? Ss 170 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 174 amino acid residues (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6 r-, a- Met Leu Pro Arg L? u Ser Phe Aon Aßn Val Ber Trp Thr Leu Lou Tyr 1 5 10 15 Tyr Lou Phe Il? Ph? Gln Val? Rg Cly slu At-p Ger Gln Lyp? The Val 20 25 30 Pro Ser Thr Arg Thr Ser Cys Pro K9t sly Ser Lyo? The Tyr Aro; Ear 35 40 45 Tyr Cyß Tyr Tbr L? U Val Thr Thr Leu yB Ser Trp Phe sln Al »? ßp 50 55 60 Leu Ala Cy? sln Lya Arg Pro be sly Hip Leu Val Ser He Leu Ser 65 70 75 eo Gly Cly Glu Wing Being Phe Val Being Ser Leu Val Thr Gly? Rg Val? On 85 90 95 Aan Aan Qln ABp Ha Trp He Trp Leu Hiß Aßp Pro Thr Met Cly Gln 100 105 110 Gln Pro Apn Cly Cly Cly Trp slu Trp Sor Aon Ser Aop Val Lou? Cn 115 120 325 Tyr Leu Aon Trp? Op Gly Aßp Pro Ser Ser Thr Val A? N? Rg Gly Aon 130 135 140 Cy? Cly S? R Leu Thr? Thr Ser Clu Ph? Leu Lyp Trp Cly Anp Hl? 145 150 155 160 Hio Cyo Aop Val Glu Leu Pro Phe Val Cya L or Pho Lyß cln 165 170 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 165 amino acid residues (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: VA i Met Thr Arg Aon Lya Tyr Phe He Leu Leu be Cyß Leu MeV. Val Leu 1 5 10 15 Sßr Pro Sßr Gln Gly Qln Glu Wing Glu Glu? Op Lou Pro Ser? La? Rg 20 25 30 He Thr Cya Pro Glu Gly Ser Aan? Tyr Sor Ser Tyr Cyo Tyr Tyr 35 40 45 Ph? Met slu Aap HID Leu Ser Trp Wing Glu Wing Aop Leu Phß Cyß Oln 50 55 60 Aßn Het Aan Ser Cly Tyr Leu Val Ser Val Leu Ser Cln Ala Olu Cly 65 70 75 80 Aßn Phe Lou Wing Sßr Leu lio Lyn Clu Ber Gly Thr Thr Wing Wing Even 85 90 95 V? .1 Trp Ilß Gly Leu His Aap Pro Lyo Aon Aon? Rg Arg Trp Hit. Trp 100 105 110 Ser Ser Gly Ser Leu Phß Leu Tyr Lys Ser Trp? Op Thr cly T r Pro 115 120 125 Aßn AB? Ser Aon? Rg Cly Tyr Cyo Val Ger Val Thr Ser? On Sister Gly 130 135 140 Tyr Lyß Lyß Trp Arg Aßp Apn Ser Cyß Aop Wing Gln Leu Ser Phe Val 145 150 155 160 V .7H

Claims (98)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A preparation of a mammalian INGAP protein substantially free of other mammalian proteins. The preparation according to claim 1, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO: 2. 3. A preparation of a polypeptide comprising a sequence of at least 15 consecutive amino acids of an INGAP protein of mammal. 4. The preparation according to claim 3, wherein the polypeptide is a fusion of the sequence with a second polypeptide derived from a second protein. The preparation according to claim 3, wherein the polypeptide is conjugated with a second polypeptide. The preparation according to claim 3, wherein the polypeptide is conjugated with a solid support. The preparation according to claim 3, wherein the polypeptide has a biological activity of the mammalian INGAP protein. 8. The preparation according to claim 7, in f. • '! where the biological activity is the ability to stimulate the growth and proliferation of pancreatic duct cells. The preparation according to claim 3, wherein the polypeptide comprises amino acids from # 103 to # 122 of the mammalian INGAP protein as set forth in SEQ ID NO: 2. The preparation according to claim 3, wherein the polypeptide comprises at least 130 consecutive amino acids of the mammalian INGAP protein. 11. An isolated DNA molecule that encodes a mammalian INGAP protein. The DNA molecule according to claim 11, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO: 2. 13. The DNA molecule according to claim 11, wherein the INGAP protein has the nucleotide sequence. shown in SEQ ID NO: 1. 14. A vector comprising the DNA of claim 11. 15. The vector according to claim 14, further comprises the expression of the control sequences, whereby the DNA is expressed in a cell Guest. 16. The vector according to claim 15, comprising an EBNA His plasmid. l'A 'I P 17. A host cell transformed with the DNA of claim 11. 18. A host cell transformed with vector of claim 14. 19. The host cell according to claim 17, which is a cos7 cell of the African Green Monkey kidney. 20. A preparation of a mammalian INGAP protein prepared by the process of: induce the expression of INGAP protein by mammalian pancreatic cells by wrapping them with cellophane; Y purify the INGAP protein from mammalian pancreatic cells induced. 21. A nucleotide probe comprising at least 20 contiguous nucleotides of a mammalian INGAP gene. 22. The nucleotide probe according to claim 21, wherein the mammalian INGAP gene has the sequence shown in SEQ ID NO: 1. 23. The nucleotide probe according to claim 21, wherein the probe is labeled with a detectable entity. . 24. A DNA molecule comprising at least 20 contiguous nucleotides of a mammalian INGAP gene. 25. The DNA molecule according to claim 24, wherein the mammalian INGAP gene has the sequence E '< 17H shown in SEQ ID NO: 1. 26. The DNA molecule according to claim 24, wherein the molecule is labeled with a detectable entity. 27. A preparation of an INGAP protein from a purified mammal substantially comprising the other mammalian proteins wherein the INGAP protein can be induced by wrapping the pancreas of the mammal with cellophane. 28. A method for the isolation of an INGAP gene from a mammal, comprising: hybridizing one or more oligonucleotides comprising at least contiguous nucleotides of the sequence shown in SEQ ID NO: 1 with genomic DNA or cDNA of the mammal; identify the DNA molecules of the DNA or of the genomic cDNA that hybridize with one or more oligonucleotides. 29. The method according to claim 28, wherein two oligonucleotides are hybridized with the DNA or with the genomic cDNA of the mammal and the oligonucleotides are used as primers in a polymerase chain reaction (PCR) to synthesize the INGAP nucleotides of the mammal. 30. The method according to claim 28, wherein one or more of the oligonucleotides are labeled. 31. The method according to claim 28, wherein the genomic DNA or cDNA of the mammal used in the hybridization step is in the form of a library of molecular clones. 32. An isolated cDNA molecule obtained by the process of: hybridizing one or more oligonucleotides comprising at least 10 contiguous nucleotides of the sequence shown in SEQ ID NO: 1 with the DNA or with the genomic cDNA of the mammal; identifying, from the DNA or the genomic cDNA, the DNA molecules that hybridize with one or more oligonucleotides. 33. An antibody preparation that is specifically immunoreactive with a mammalian INGAP protein. 34. The antibody preparation according to claim 33, wherein the mammalian INGAP protein has an amino acid sequence such as that shown in SEQ ID NO: 2. The antibody preparation according to claim 33, which is polyclonal. 36. The antibody preparation according to claim 33, which is monoclonal. 37. The antibody preparation according to claim 33, comprising antibodies that are bound to a solid phase. 38. A hybridoma that produces antibodies that are specifically immunoreactive with a mammalian INGAP protein. 39. A method for producing a mammalian INGAP protein, comprising the steps of: providing a host cell according to claim 17; culturing the host cell in a nutrient medium such that the INGAP protein is expressed, and - harvesting the INGAP protein from the host cells or from the nutrient medium. 40. A method for producing a mammalian INGAP protein, comprising the steps of: providing a host cell comprising the DNA molecule according to claim 11; culturing the host cell in a nutrient medium so that the mammalian INGAP protein is expressed; and harvesting the mammalian INGAP protein from the host cells or the nutrient medium. 41. A method for the treatment of diabetic mammals, comprising: administering to a diabetic mammal a therapeutically effective amount of an INGAP protein for f? > < stimulate the growth of islet cells. 42. The method according to claim 41, wherein the mammal has insulin-dependent diabetes mellitus. 43. The method according to claim 41, wherein the mammal has diabetes mellitus not dependent on insulin. 44. A method for the growth of pancreatic islet cells in culture, comprising: supplying an INGAP protein to a culture medium for the growth of pancreatic islet cells; and growing the islet cells in the culture medium comprising INGAP protein. 45. A method for increasing the life of pancreatic islet cells encapsulated in a polycarbonate shell, comprising: adding an INGAP protein in encapsulated pancreatic islet cells in an amount sufficient to increase the survival rate or survival time of pancreatic islet cells. 46. A method for increasing the number of pancreatic islet cells in a mammal, comprising: administering to the pancreas of a mammal a DNA molecule encoding an INGAP protein. 47. The method according to claim 46, wherein the DNA molecule has the sequence shown in SEQ ID NO: 1. 48. The method according to claim 46, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO. : 2. 49. A method for increasing the number of pancreatic islet cells in a mammal, comprising: administering an INGAP protein to the pancreas of a mammal. 50. The method according to claim 49, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO: 2. 51. A transgenic mammal comprising an INGAP gene of a second mammal. 52. The transgenic mammal according to claim 51, wherein the INGAP gene has the sequence shown in SEQ ID NO: 1. 53. The transgenic mammal according to claim 51, wherein the INGAP gene is expressed at a level higher than that of any endogenous INGAP gene of the mammal. 54. A non-human mammal that has been manipulated l'.l / '! genetically to contain an insertion or deletion mutation of an INGAP gene of the mammal. 55. A method for identifying individual mammals at risk for diabetes, comprising: identifying a mutation in an INGAP gene from a sample of an individual mammal, the mutation causing a structural abnormality in an INGAP protein encoded by the gene or causing a regulatory defect that leads to a diminished or obliterated expression of the INGAP gene. 56. The method according to claim 55, wherein the sample is a blood sample. 57. The method according to claim 55, wherein the sample is amniotic fluid. 58. The method according to claim 55, wherein the sample is chorionic villus. 59. The method according to claim 55, wherein the sample comes from a blastocyst. 60. The method according to claim 55, wherein the sample is pancreatic cells. 61. A method for detecting INGAP protein in a sample from a mammal, comprising: contacting the sample with an antibody preparation according to claim 33. i. 62. The method according to claim 61, wherein a predetermined amount of a polypeptide comprising at least 15 consecutive amino acids of a mammalian INGAP protein is also contacted with the sample. 63. The method according to claim 62, wherein the polypeptide is labeled in detectable form. 64. The method according to claim 61, wherein the antibody preparation comprises antibodies that are attached to a solid support. 65. The method according to claim 62, wherein the antibody preparation comprises antibodies that are attached to a solid support. 66. The method according to claim 65, further comprising the step of: detecting the labeled polypeptide that is not bound to the solid support. 67. A method for the treatment of islet cells isolated from a mammal to prevent apoptosis of cells, comprising: contacting islet cells isolated from a mammal with a preparation of a mammalian INGAP protein, substantially purified from other mammalian proteins, "in an amount sufficient to increase the survival rate of the cells of . 1. Isolated islet. 68. A method for the treatment of a mammal that receives an islet cell transplant, comprising: administering a preparation of a mammalian INGAP protein to a mammal receiving an islet cell transplant, wherein the administration paeo It is done before, during or after the transplant. 69. The method according to claim 68, wherein the step of administering is carried out intravenously. 70. The method according to claim 68, wherein the administration step is effected by local perfusion at the site of the transplant. 71. The method according to claim 68, wherein the step of administration is via the portal vein. 72. The method according to claim 71, wherein the islet cells are concomitantly transplanted via the portal vein. 73. A method for inducing differentiation of cell progenitors | l, comprising: contacting a culture of pancreatic duct cells comprising β cell progenitors with a preparation of a mammalian INGAP protein i. . H substantially free of other mammalian proteins, to induce the differentiation of the progenitors of the β-74 cell. A method for the treatment of a mammal with pancreatic endocrine failure or problem, comprising: contacting a duct cell preparation pancreatic comprising β-cell progenitors isolated from a mammal afflicted with pancreatic endocrine failure or problem with a preparation of a mammalian INGAP protein subetanially free from other mammalian proteins, to induce differentiation of β-cell progenitors; and transplant in an autologous way the lae cells of the pancreatic duct within the mammal. 75. An antisense construct of a mammalian INGAP gene comprising: a promoter, a terminator and a nucleotide sequence consisting of a mammalian INGAP gene, the nucleotide sequence between the promoter and the terminator, the nucleotide sequence is inverted with respect to the promoter, whereby with the expression of the promoter an mRNA complementary to the INGAP mRNA of the native mammal is produced. 76. A method for the treatment of nesidioblastosis, which comprises: I '4 administer to a mammal with nesidioblastosis, an antisense construct according to claim 75, thereby inhibiting the overgrowth of the mammalian β cells. 77. A kit for detecting a mammalian INGAP protein in a sample from the mammal, comprising: an antibody preparation that is specifically immunoreactive with a mammalian INGAP protein; a polypeptide comprising a sequence of at least 15 consecutive amino acids of a mammalian INGAP protein. 78. The kit according to claim 77, wherein the polypeptide is labeled in detectable form. 79. The kit according to claim 77, wherein the antibody preparation comprises antibodies that are bound to a solid support. 80. A pharmaceutical composition for the treatment of pancreatic insufficiency, comprising: a mammalian INGAP protein in a pharmaceutically acceptable diluent or carrier. 81. The pharmaceutical composition according to claim 80, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO: 2. 82. A pharmaceutical composition comprising: a preparation of a polypeptide comprising a sequence of at least 15 amino acids coneecutivee of a mammalian INGAP protein and a pharmaceutically acceptable diluent or carrier. 83. The pharmaceutical composition according to claim 82, wherein the polypeptide is a fusion of the sequence with a second polypeptide derived from a second protein. 84. The pharmaceutical composition according to claim 82, wherein the polypeptide is conjugated with a second polypeptide. 85. The pharmaceutical composition according to claim 82, wherein the polypeptide has the biological activity of the mammalian INGAP protein. 86. The preparation according to claim 85, wherein the biological activity is the ability to stimulate the pancreatic duct cells to grow and proliferate. 87. The pharmaceutical composition according to claim 82, wherein the polypeptide comprises amino acids from # 103 to # 122 of the mammalian INGAP protein as shown in SEQ ID NO: 2. 88. The pharmaceutical composition according to claim 82, in where the polypeptide comprises at l. at least 130 consecutive amino acids of the mammalian INGAP protein. 89. A method for the identification of cell progenitors, comprising: contacting a population of pancreatic duct cells with a mammalian INGAP protein preparation; and detecting cells from the population to which INGAP specifically binds. 90. The method according to claim 89, wherein the INGAP protein is labeled in detectable form. 91. The method according to claim 89, wherein the INGAP protein is immobilized on a solid phase. 92. The preparation according to claim 1, wherein the INGAP protein is human and comprises amino acid sequences 1 to 83 and 124 to 174 as set forth in SEQ ID NO: 2. 93. The preparation according to claim 1, wherein the INGAP protein is human and comprises, in an N-terminal to C-terminal orientation: amino acids 1 to 83 in SEQ ID NO: 2, 40 amino acids and amino acids 124 to 174 in SEQ ID NO: 2. 94. DNA molecule according to claim 11, wherein the INGAP protein is from human. I -1 95. The DNA molecule according to claim 94, wherein the INGAP protein comprises amino acid sequences 1 to 83 and 124 to 174 in SEQ ID NO: 2. 96. The DNA molecule according to claim 94, wherein the pipetine INGAP comprises, in an N-terminal to C-terminal orientation, amino acids 1 to 83 in SEQ ID NO: 2, 40 amino acids and amino acids 124 to 174 in SEQ ID NO: 2. 97. The DNA molecule according to claim 24, encoding an amino acid sequence selected from those of amino acids 1 to 83 and 124 to 174 in SEQ ID NO: 2. 98. The DNA molecule according to claim 11, comprising nucleotides 4 to 268 and 389 to 629 of SEQ ID NO: 2.