60%, coinciding with the proliferation of a mono-layer of astrocytes. In contrast, under conditions in which the proliferation of astrocytes was inhibited / dopaminergic neurons, but not GABAergic, were almost eliminated from the cultures for 5 days in vi tro. These results demonstrate the importance of homotypically derived astrocytes for the survival and development of adjacent dopaminergic neurons, and suggest that mesencephalic astrocytes are probably a source of physiological paracrine neurotrophic factor for mesencephalic dopaminergic neurons. The repeated demonstration that astrocytes secrete molecules that promote neuronal survival has made astrocytes a focus in the search for therapies to treat neurodegenerative diseases. Many laboratories have attempted to isolate neurotrophic factors derived from astrocytes, but have been hampered by a major technical problem: serum is an essential component of the medium for optimal growth of primary astrocytes in culture, although the presence of serum interferes with subsequent purification of the factors secreted in the conditioned medium. Thus, there is a need to identify and purify new neurotrophic factors and identify new methods to produce conditioned medium that is compatible with protein isolation techniques.
A cell line similar to spontaneously immortalized astrocytes type-1, known as the ventral mesencephalic cell line-1 (VMCL-1), has been isolated. This cell line, deposited in the American type culture collection (ATCC, Manassas, Virginia, United States, ATCC Access number:, date of deposit, September 18, 2000), is derived from the ventral mesencephalon and retains the characteristics of astrocytes primary type 1, but grows robustly in serum-free medium. The conditioned medium prepared from these cells contains one or more neuronal survival factors that increase the survival of mesencephalic dopaminergic neurons at least 3 times, and also promotes their development. The potency of this neurotrophic activity and its low degree of toxicity on dopaminergic neurons in vi tro are distinctive features of the activity of VMCL-1 CM. Furthermore, using size fractionation techniques, we have identified activities that elute at approximately 14-16 kiloDaltons, 18-21 kiloDaltons, and 25-35 kiloDaltons. The immortalized cell line VMCL-1 does not require serum for its growth and thus allows us to identify the polypeptides that promote neuronal survival VMCL-1 CM. Using a multi-step purification process, we identify arginine-rich protein (which has a molecular weight of approximately 20 kiloDaltons) as a protein that is co-purified with an activity that promotes neuronal survival. As the protein and the activity are copurified through five purification steps, we conclude that this protein is one of the factors in the CMVL-1 CM that has the activity that promotes the desired neuronal survival. In accordance with the foregoing, in general, the invention presents methods for increasing the survival of neurons (eg, dopaminergic neurons), as well as new polypeptides that exhibit this activity that promotes neuronal survival. In a first aspect, the invention features a pharmaceutical composition that includes, as an active polypeptide, a protein substantially rich in arginine, and a pharmaceutically acceptable carrier. In a preferred embodiment, the arginine-rich protein is a protein rich in human arginine (SEQ ID NO: 1). In a second aspect, the invention features a substantially pure polypeptide having a molecular weight of about 14-16 kiloDaltons which increases the survival of dopaminergic neurons. In a third aspect, the invention features a substantially pure polypeptide having a molecular weight of about 18-21 kiloDaltons which increases the survival of dopaminergic neurons. In a fourth aspect, the invention features a substantially pure polypeptide having a molecular weight of about 25-35 kiloDaltons which increases the survival of dopaminergic neurons. The polypeptides of the present invention can be obtained from a glial cell line, such as VMCL-1 or another immortalized type-1 astrocyte cell line. In preferred embodiments of the first, second, third or fourth aspect, the survival of dopaminergic neurons increases at least three times. More preferably, survival increases at least four times, although more preferably, survival increases at least five times. In another aspect, the invention presents a method for increasing dopaminergic neuronal survival. The method includes contacting a dopaminergic neuron (either in vi tro or in vivo) with a polypeptide of the first, second, third or fourth aspect. A preferred polypeptide is a protein rich in human arginine. Preferably, the survival of dopaminergic neurons increases at least three times, more preferably at least four times, and much more preferably at least five times. In another aspect, the invention features a method for culturing dopaminergic neurons for transplantation, including the step of culturing the neurons, or progenitor cells thereof, with an effective amount of a first, second, third, or fourth aspect polypeptide. As before, a preferred polypeptide is the protein rich in human arginine. In preferred embodiments, the amount is sufficient to increase the survival of dopaminergic neurons by at least three times, at least four times, or up to at least five times. In still another aspect, the invention features a method for treating a patient having a disease or condition of the nervous system, this method includes the step of administering to the patient an amount that promotes the survival of a protein rich in substantially purified arginine. In still another aspect, the invention presents another method for preventing death of dopaminergic neuronal cells in a mammal. This method includes administering to the mammal an amount that promotes the survival of dopaminergic neurons of a protein rich in substantially purified arginine. A preferred mammal is a human. The invention also features a method of transplanting cells into the nervous system of a mammal, including (i) transplanting cells into the nervous system of the mammal; and (ii) administering an amount that promotes the dopaminergic neuronal survival of the arginine-rich protein (e.g., human arginine-rich protein) to the mammal (e.g., a human) at a four-hour time interval before transplanting the cells up to four hours after cell transplantation. In preferred embodiments, the time interval is two hours before transplantation of the cells at two hours after the transplantation of the cells.The invention presents another method of transplanting cells into the nervous system of a mammal. In this method, the cells are contacted with arginine-rich protein; and then they are transplanted into the nervous system of the mammal. Preferably, these two steps are performed within four hours of each other. In yet another aspect, the invention features a method for the preparation of a dopaminergic neuronal survival-promoting polypeptide of the present invention, including culturing an immortalized type-1 astrocyte cell line under conditions that allow expression of the polypeptide. In yet another aspect, the invention features a substantially pure composition that includes a polypeptide that increases the survival of dopaminergic neurons, the polypeptide having a molecular weight of about 14-16 kiloDal-tons, about 18-21 kiloDaltons, or about 25-35 kiloDaltons. Methods of treating diseases and conditions using the polypeptides or compositions of the invention are also characteristic of the invention. For example, a method of treating a disease or condition of the nervous system (e.g., Parkinson's disease) can be effected with the described polypeptides. The invention also provides a method for preventing dopaminergic neuronal cell death by administering an effective amount of the polypeptide of the invention. This medicine is made by administering the polypeptide with a pharmaceutically acceptable carrier. The invention features the use of a polypeptide of the first, second, third or fourth aspect in the manufacture of a medicament. The invention further features the use of a polypeptide as defined herein: (1) for immunizing a mammal to produce anti-bodies, which optionally can be used for therapeutic or diagnostic purposes; (2) in a competitive assay to identify or quantify molecules that have receptor binding characteristics corresponding to those of the polypeptide; (3) to contact a sample with a polypeptide, as mentioned above, together with a receptor capable of binding specifically to the polypeptide for the purpose of detecting competitive inhibition of binding to the polypeptide; and (4) in an affinity isolation process, optionally affinity chromatography, for the separation of a corresponding receptor. As mentioned above, the invention provides, for mammalian sources, novel dopaminergic neuronal survival factors (e.g., arginine-rich protein) that are distinguishable from known factors. These factors promote the survival of dopaminergic neurons. The -Yes-invention also provides processes for the preparation of these factors, and a method to define the activity of these and other factors. The therapeutic application of the factors is a more significant aspect of the invention. In other aspects, the invention features a polypeptide that increases the survival of dopaminergic neurons, the polypeptide having a molecular weight of about 14-16 kiloDaltons or 25-35 kiloDaltons (relative to proteins of known molecular weights, ranging from 15-102 kiloDaltons, run under the same conditions), as determined using a CL-6B column of heparin-sepharose (Sigma Chemicals, St. Louis, Missouri, United States), and which has an activity that promotes survival for dopaminergic neurons. It will be appreciated that the aforementioned molecular weight range limits are not accurate, and are not subject to slight variations depending on the source of the particular polypeptide factor. A variation of approximately 10 percent for example would not be impossible for material from another source. In another aspect, the invention features a pharmaceutical formulation that includes a polypeptide of the present invention formulated for pharmaceutical use, optionally together with an acceptable diluent., vehicle or excipient and / or in the form of a unit dose. By using the factors of the invention, conventional pharmaceutical practice can be employed to provide convenient formulations or compositions. For example, it is preferred that any viral pathogens that may be present with the substantially pure polypeptide be removed or inactivated, and that similar prtive measures be taken to remove any toxic compound that is present with the substantially pure polypeptide. In one embodiment, the pharmaceutical formulation includes cells (e.g., dopaminergic neurons or their progenitors) for transplantation. In another aspect, the invention features a method of transplanting cells (e.g., dopaminergic neurons or their progenitors) into the nervous system of a mammal. The method includes administering a polypeptide or composition of the present invention, in a pharmaceutically acceptable carrier to the mammal before, during, or after transplantation of the cells. It is preferred that the polypeptide or composition be administered to the mammal at a time interval from four hours before the transplant to four hours after the transplant. More preferably, the time interval is two hours before the transplant two hours after the transplant. It is understood that the polypeptide or composition has to be present for the entire time interval to prt or decrease cell death. In a related aspect, the invention presents another method of transplanting cells into the nervous system of a mammal. The method includes contacting the cells to be transplanted with a polypeptide or composition of the present invention, in a pharmaceutically acceptable carrier prior to cell transplantation. It is preferred that the cells to be transplanted are contacted with the polypeptide or compound within four hours of the transplant, and, more preferably within two hours of the transplant. It is understood that the polypeptide or composition does not have to be present for the entire time in order to avoid or decrease cell death after transplantation. Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, the formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. Methods well known in the art for making formulations were found in, for example, Remington: The Science and Practice of Pharmacy, (19th ed.) Editors A.R. Gennaro AR. , 1995, Mack Publishing Company, Easton, Pennsylvania, United States. Formulations for parenteral administration may contain, for example, as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biodegradable, biocompatible lactide polymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The formulations for inhalation may contain as excipients, for example, lactose, or they may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or they may be oily solutions for administration in the form of nasal drops. , or as a gel to be applied intranasally. The present factors can be used as the sole active agents, or they can be used in combination with other active ingredients, for example, other growth factors that could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors. The concentration of the present factors in the formulations of the invention will vary depending on several points, including the dosage to be administered, and the route of administration. In general terms, the factors of this invention can be provided in an aqueous physiological regulated solution containing approximately 0.1 to 10 weight / volume of polypeptide for parenteral administration. The general dose ranges are from about 1 mg / kg to about 1 g / kg of body weight per day; A preferred dose range is from about 0.01 mg / kg to 100 mg / kg of body weight per day. The preferred dose to be administered probably depends on the type and degree of progress of the pathophysiological condition being attacked, the overall health of the patient, the make-up of the formulation, and the route of administration. As indicated above, the dopaminergic neurons-to a large extent, are prevented from drying out in the presence of the factors of the invention. Dopaminergic neurons of the mesencephalon die in patients who have Parkinson's disease. The invention thus provides a treatment of Parkinson's disease. In addition, the use of the present factors in the treatment of diseases or diseases of the nervous system in which the loss of dopaminergic neurons is present or anticipated is included in the invention. The invention also presents methods of analysis to identify factors to enhance or mimic the activity that promotes neuronal survival rich in arginine. In these assay methods for enhancers, the ability of candidate compounds to increase the expression of arginine-rich proteins, biological activity or stability using standard techniques is tested. A candidate compound that binds with arginine-rich protein can act as a potentiating agent. Alternatively, a mimetic compound (i.e., a compound that binds to the arginine-rich protein receptor) is capable of acting in the absence of the arginine-rich protein. By "substantially pure" is meant that a polypeptide (e.g., the arginine-rich protein) has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60 percent, by weight, and free of naturally occurring organic proteins and molecules with which it is naturally associated. Preferably, the polypeptide is an arginine-rich protein that is at least 75 percent, more preferably at least 90 percent, and much more preferably at least 99 percent, by weight, pure. A protein rich in substantially pure arginine can be obtained, for example, by extracting a natural source (e.g., a neuronal cell), by expressing a recombinant nucleic acid encoding an arginine-rich protein, or by chemically synthesizing the protein. The purity can be measured by any suitable method, for example, by column chromatography, polyacrylamide gel electrophoresis, or high performance liquid chromatography analysis. A polypeptide is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a polypeptide that is synthesized or chemically produced in a cell system different from the cell from which it naturally originates will be substantially free of its naturally associated components. In accordance with the foregoing, substantially pure polypeptides include those that occur naturally in eukaryotic organisms but are synthesized in E. coli or other prokaryotes. By "polypeptide" or "protein" is meant any chain of more than two amino acids, independently of post-translational modification such as glycosylation or phosphorylation. An arginine-rich protein that is part of the invention includes a protein having an activity that promotes dopaminergic neuronal survival and encoded by a nucleic acid that hybridizes very strongly to the cDNA encoding the human arginine-rich protein. A preferred arginine-rich protein is represented by the amino acid sequence of SEQ ID NO: 1. The nucleic acids that are a part of the invention include those nucleic acids that encode proteins having activity that promotes the dopaminergic neuronal test signal and that hybridizes very strongly to one of the cDNA chains encoding the human arginine-rich protein (SEQ ID NO: 5). A preferred nucleic acid is represented by the nucleotide sequence of SEQ ID NO: 5. By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 50 percent, preferably 85 percent, more preferably 90 percent, and much more preferably 95 percent identification with a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and much more preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (eg, sequence analysis software package from the genetics computer group, University of Wisconsin Biotechnology Center, 1710 University Avenue, adison, Wisconsin 53705, United States). This software program compares similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine and phenylalanine, tyrosine. By "high stringency condition" is meant hybridization in 2X SSC at 40 ° C with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see F. Ausubel et al., Current Protocole in Molecular Biology, pages 6.3.1-6.3.6, John iley & Sons, New York, 1994, incorporated herein by reference. By "polypeptide" or "factor" is meant a molecule that has an activity that promotes the test signal (or, conversely, prevents death) of dopaminergic neurons in a standard cell survival assay. The compounds of the present invention have a molecular weight of about 14-16 kiloDaltons, about 18-21 kiloDaltons, or, alternatively, about 25-35 kiloDaltons. Specifically excluded from the polypeptides of the invention are glial cell-derived neurotrophic factor (GDNF) (Lin et al., Science 260: 1130-1132, 1993), neurturin (Kotzbauer et al., Nature 384: 467-470, 1996), persefin (Millbrandt et al., Neuron 20: 245-253, 1998), and artemin (Baloh et al., Neuron 21: 1291-1302, 1998). By "composition" is meant a collection of polypeptides, including a polypeptide of the present invention. By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the polypeptide with which it is administered. A pharmaceutically acceptable exemplary vehicle is physiological saline. Other physiologically acceptable carriers and their formulations are known to a person skilled in the art and are described, for example, in Remin ton: The Science and Practice of Pharmacy, (19th edition) editor A.R. Gennaro AR. , 1995, Mack Publishing Company, Easton, Pennsylvania, United States. It will be understood that viral pathogens and toxic compounds that are inadvertently included by a polypeptide or composition of the present invention can be inactivated or removed using any method known in the art. A compound having "activity that promotes dopaminergic neuronal survival" means the presence of the compound increases the survival of dopaminergic neurons at least twice in a neuronal survival assay
(such as that described herein) in relation to the survival of dopaminergic neurons in the absence of the compound. Preferably, the increase in the survival of dopaminergic neurons is at least three times, more preferably at least four times, and much more preferably at least five times. The assay can be an in vi tro assay or an in vivo assay. Preferably, the assay is an in vi tro assay
(See the section entitled "cell viability assay", infra). The present invention provides new methods and reagents for the prevention of neuronal cell death. The invention also provides pharmaceutical compositions for the treatment of neurological diseases or conditions of which aberrant neuronal cell death is one of the causes.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Detailed Description of the Invention It has been discovered that a cell line of mesencephalic origin (termed "VMCL-1") secretes a factor that, in turn, promotes the differentiation and survival of dopaminergic neurons. This cell line grows robustly in a serum-free medium. Moreover, the conditioned medium prepared from these cells contains one or more neuronal survival factors that increase the survival of the mesencephalic neurodegenerative neurons by at least three times, and promotes their development as well. A protein that we identified as an arginine-rich protein was purified from the VMCL-1 cell line. We purified this protein as follows. A volume of three liters of conditioned medium of VMCL-1 was prepared and subjected to five sequential steps of column chromatography. In each purification step, each column fraction was examined to determine the biological activity in the bioassay referred to above. An estimate of the effect of each fraction on neuronal survival was made at 24-hour intervals, over a period of five days, and scored on a scale of 1 to 10. After the fifth purification step, the biologically active fraction and a inactive adjacent fraction were analyzed by SDS-PAGE. The results of the SDS-PAGE analysis revealed a distinctive protein band in the range of 20 kiloDaltons in the active fraction. The "active" band was separated and subjected to tryptic digestion, and the molecular mass and sequence of each background peptide was determined by mass spectrometric analysis. The following two peptide sequences were identified: DVTFSPATIE (SEQ ID NO: 3) and QIDLSTVDL (SEQ ID NO: 4). A search of the database identified a protein match rich in human arginine (SEQ ID NO: 1) and its mouse ortholog (SEQ ID NO: 2). The predicted protein encoded by the mouse EST sequence is approximately 95 percent identical to the predicted human protein. A search of the rat EST database revealed two sequences, one (dbEST Id: 4408547; EST name: EST348489) which has a significant homology at the amino acid level with the human and mouse proteins. The full-length rat sequence is not in the GenBank database. Thus, the arginine-rich protein is useful as a neurotrophic factor for the treatment of a neurodegenerative disease and for improving neuronal survival during or after transplantation in a human. The protein rich in arginine can also be used to improve the production of in vitro neurons for transplantation. In another use, the arginine-rich protein allows the identification of compounds that modulate or mimic its activity that promotes dopaminergic neuronal survival. The protein can also be used to identify your cognate receptor. Each of these uses is described in more detail later. Identification of molecules that modulate the biological activity of arginine-rich protein The effect of candidate molecules on the arginine-rich protein mediated regulation of neuronal survival can be measured at the level of translation using standard protein detection techniques, such as Western blotting or immunoprecipitation with a specific anti-body protein rich in arginine. The compounds that modulate the level of the arginine-rich protein can be purified, or substantially purified, or they can be a component of a mixture of compounds such as an extract or a supernatant obtained from cells (Ausubel et al., Supra). In an assay of a mixture of compounds, the expression of the arginine-rich protein is measured in cells progressively administered to smaller subsets of the compound deposit (eg, produced by standard purification techniques such as HPLC or FPLC) until a only compound or a minimum number of effective compounds are shown to express the arginine-rich protein. The compounds can also be directly selected for their ability to modulate neuronal survival mediated by arginine-rich protein. In this approach, the amount of neuronal survival in the presence of a candidate compound is compared to the amount of neuronal survival in its absence, under equivalent conditions. Again, the selection may begin with a deposit of candidate compounds, from which one or more useful modulatory compounds are isolated in a staggered manner. The activity that promotes survival can be measured by any standard assay. Another method to detect compounds that modulate the activity of arginine-rich protein is to select compounds that physically interact with arginine-rich protein. These compounds can be detected by adapting the interaction trap expression systems known in the art. These systems detect protein interactions using a transcription activation assay and are generally described by Gyuris et al. (Cell 75: 791-803, 1993) and Field et al. (Nature 340: 245-246, 1989). Alternatively, the arginine-rich protein or biologically active fragments thereof can be labeled with Bolton-Hunter 125I reagent (Bolton et al., Biochem. J. 133: 529, 1973). The candidate molecules previously accommodated in the wells of a multi-well tray are incubated with labeled arginine-rich protein, washed and any well with labeled arginine-rich protein complex is examined. The data obtained using different concentrations of arginine-rich protein are used to calculate values of the number, affinity, and association of arginine-rich protein with candidate molecules. Compounds of molecules that function as modulators of the activity that promotes neuronal survival of the arginine-rich protein can include peptide and non-peptide molecules such as those present in cell extracts, mammalian serum, or growth medium in which the mammalian cells have been cultured. A molecule that modulates the expression of arginine-rich protein or the biological activity mediated by arginine-rich protein such that there is an increase in the survival of neuronal cells is considered useful in the invention; such a molecule can be used, for example, as a therapeutic agent, as described below. Therapy The discovery of proteins rich in arginine as a neurotrophic factor that promotes the survival of dopaminergic neurons allows their use for the therapeutic treatment of neurodegenerative diseases such as Parkinson's disease. To add arginine-rich protein to cells in order to prevent neuronal death, it is preferable to obtain sufficient quantities of pure recombinant arginine-rich protein from cultured cell systems that can express the protein. The preferred arginine-rich protein is protein rich in human arginine, but the arginine-rich protein derived from other animals (eg, pig, rat, mouse, dog, baboon, cow, and the like) can also be used. The administration of the protein to the affected tissue can be carried out using suitable packaging or administration systems. Alternatively, small molecule analogs can be used and administered to act as arginine-rich protein agonists and thus produce a desired physiological effect. Gene therapy is another potential therapeutic approach in which normal copies of the gene encoding the arginine-rich protein (or nucleic acid encoding the RNA sense of arginine-rich protein) are introduced into the cells to successfully produce arginine-rich protein. . The gene should be administered to those cells in a form in which it can be absorbed and encoded for sufficient proteins to provide activity that promotes effective neuronal survival. Retroviral vectors, adenoviral vectors, viral vectors associated with adenovirus, or other viral vectors with the tropism suitable for nerve cells can be used as a gene transfer delivery system for a protein arginine rich protein gene construct. Numerous vectors useful for this purpose are known in general (Miller, Human Gene Therapy 15-14, 1990, Friedman, Science 244: 1275-1281, 1989, Eglitis and Anderson, BioTechniques 6: 608-614, 1988, Tolstoshev and Anderson, Curr Opin Biotech 1: 55-61, 1990, Sharp, The Lancet 337: 1277-1278, 1991, Cornetta et al, Nuci, Acid Res. And Mol. Biol. 36: 311-322, 1987, Anderson, Science 226: 401-409, 1984, Moen, Blood Cells 17: 407-416, 1991, Miller et al., Biotech 7: 980-990, 1989, Le Gal La Salle et al., Science 259: 988-990, 1993.; and Johnson, Chest 107: 77S-83S, 1995). Retroviral vectors develop particularly well and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med. 323: 370, 1990, Anderson et al., US Pat. No. 5,399,346). Non-viral approaches can also be employed for the introduction of therapeutic DNA into the desired cells. For example, the arginine-rich protein can be introduced into a cell by lipofection (Felgner et al., Proc., Nati, Acad. Sci USA 84: 7413, 1987, Ono et al., Neurosci. Lett. 117: 259, 1990; Brigham. et al., Am. J. Med. Sci. 298: 278, 1989; Staubinger et al., Meth. Enzymol. 101: 512, 1983), asialorosonucoid-polylysine conjugation (Wu et al., J. Biol. Chem. 263: 14621 , 1988; Wu et al., J. Biol. Chem. 264: 16985, 1989); or, less preferably, microinjection in surgical conditions (Wolff et al., Science 247: 1465, 1990). Gene transfer could also be achieved using non-viral means that require infection in vi tro. This would include calcium phosphate, DEAE dextran, electroincorporation, and protoplast fusion. Liposomes can also be potentially beneficial for the administration of DNA in a cell. Although these methods are available, many of these are less efficient. Many methods for introducing vectors into cells or tissues are available and are equally suitable for in vivo use, in vi tro, and ex vivo. For ex vivo therapy, vectors can be introduced into neural stem cells taken from the patient and propagated clonally for autologous transplantation again in the same patient. Administration by transfection and liposome injections can be achieved using methods that are well known in the art. Transplanting normal genes in affected cells of a patient can also be useful therapy. In this procedure, a normal arginine-rich protein gene is transferred into neurons or glia, either exogenously or endogenously to the patient. These cells are injected into the target tissue (s). In the disclosed constructs, expression of the arginine-rich protein cDNA can be directed from any convenient promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, known highlighters for preferentially directing gene expression in nerve cells can be used to direct the expression of arginine-rich protein. The mej speakers used could include, without limitation, those that are characterized as specific to the tissue or cell in its expression. Alternatively, if a genomic clone of arginine-rich protein is used as a therapeutic construct (eg, after isolation by hybridization with the cDNA of the arginine-rich protein described herein), regulation can be mediated by regulatory sequences. of cognate or, if desired, by regulatory sequences derived from a heterologous agent, including any of the promoters or regulatory elements described above. RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the view of flanking sequences at the 5'- and / or 3'-ends of the molecule or the use of phosphorothioate or 2-or-methyl instead of the phosphodiesterase bonds within the central structure. of the molecule. This concept can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, kinasin, and wentsin, as well as acetyl, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine that are not easily recognized by endogenous endonucleases. Another therapeutic approach within the invention involves the administration of recombinant arginine-rich protein, either directly to the site of actual potential cell loss (eg, by injection) or systemically (eg, by any conventional recombinant protein delivery technique). ). A further embodiment of the invention relates to the administration of a pharmaceutical composition, a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. These pharmaceutical compositions may consist of arginine-rich protein, arginine-rich protein anti-bodies, mimetics, or arginine-rich protein agonists. The compositions can be administered alone or in combination with at least some other agent, such as a stabilizing compound, which can be administered in any sterile, pharmaceutically biocompatible vehicle, including, but not limited to, saline, regulated saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones. In one example, the arginine-rich protein is administered to a subject at the site where the cells are transplanted. Administration of arginine-rich protein can be done before or after cell transplantation. Preferably, the two steps are within approximately four hours of each other. If desired, the arginine-rich protein can be repeatedly administered to the subject at various intervals before and / or after cell transplantation. This protective administration of arginine-rich protein can occur months or even years after cell transplantation. In addition to its administration to a human or other mammal, the protein rich in arginine can also be used in culture to improve the survival of neurons during production any time before transplantation. In one example, the cells to be transplanted are to be suspended in a pharmaceutical carrier which also includes an amount that promotes the survival of arginine-rich protein. The arginine-rich protein can also be administered to culture earlier in the process (for example, when the neurons are differentiating first). It is understood that neurons do not need to be primary dopaminergic neurons. Neurons (eg, dopaminergic neurons) that differentiate, either in vitro or in vivo, from stem cells or from any other cell capable of producing neurons can be grown in the presence of arginine-rich protein during their production and maintenance . Although the protein rich in human arginine is present for use in the methods described herein, the arginine-rich protein has been identified in numerous species, including rats, mice, and cows. A person skilled in the art will recognize that the identification of arginine-rich protein from other animals can easily be performed using standard methods. Any protein that has dopaminergic neuronal survival activity and encoded by nucleic acid that hybridizes cDNA encoding the human arginine rich protein is considered part of the invention. Diagnosis Anti-bodies that specifically bind with arginine-rich protein can be used for diagnosis of conditions or diseases characterized by alterations in arginine-rich protein levels, or in assays to monitor patients who are being treated with rich protein in arginine. Anti-bodies useful for the purpose of diagnosis can be prepared in the same manner as those described above for therapy. Diagnostic assays for arginine-rich protein include methods that use the anti-body and a label to detect the arginine-rich protein in human body fluids or cell or tissue extracts. Anti-bodies can be used with or without modification, and can be labeled by joining them, either covalently or non-covalently with a reporter molecule. A wide variety of reporter molecules known in the art can be used, several of which are described herein. A variety of protocols including ELISA, RIA, and FACS are known in the art to measure protein rich in arginine and provide a basis for diagnosing altered or abnormal levels of arginine-rich protein expression. Normal or standard values of the expression of arginine-rich proteins are established by combining body fluids or extracts from cells taken from normal mammalian subjects, preferably humans, with anti-body for arginine-rich protein under conditions suitable for complex formation. The amount of standard complex formation can be quantified by several methods, but preferably by photometric means. The amounts of arginine-rich protein expressed in subject, control and disease samples from biopsy tissues are compared to standard values. The deviation between the standard values and the subject establish the parameters to diagnose the disease. Nucleic acid sequences encoding arginine-rich protein can also be used for diagnostic purposes. The nucleic acid sequence that can be used includes RNA and anti-sense DNA molecules, and oligonucleotide sequences. Nucleic acid sequences can be used to detect and quantify the expression of genes in biopsy tissues in which the expression of arginine-rich protein can be correlated with the disease. The diagnostic test can be used to distinguish between absence, presence, and surplus expression of arginine-rich protein, and to monitor the regulation of arginine-rich protein levels during therapeutic intervention. The nucleic acid sequences encoding the arginine-rich protein can be used for the diagnosis of conditions or diseases that are associated with the altered expression of arginine-rich protein. The nucleic acid sequences encoding the arginine-rich protein can be used in Southern or Northern analysis, spot spotting, or other membrane-based technologies; in polymerase chain reaction technologies; or dipstick, pIN, ELISA or chip assays using fluids or tissues from patient biopsies to detect the expression of altered arginine-rich protein. These qualitative or quantitative methods are well known in the field. The nucleotide sequences encoding arginine-rich proteins can be labeled by standard methods, and added to a sample of fluid or tissue from a patient under conditions suitable for the formation of hybridization complexes. After a convenient incubation period, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signals in the sample that was biopsied or extracted is significantly altered from that of a comparable control sample, the nucleotide sequences have been hybridized with nucleotide sequences in the sample, and the presence of Altered nucleotide sequences that encode arginine-rich protein in the sample indicates the presence of the associated disease. These assays can also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to supervise the treatment of an individual patient. In order to provide a basis for the diagnosis of the disease associated with the altered expression of arginine-rich protein, a normal or standard profile for stable expression. This can be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or fragment thereof, which encodes arginine-rich protein, under conditions suitable for hybridization or amplification. Standard hybridization can be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. The standard values obtained from the normal samples can be compared with the values obtained from patient samples that are symptomatic for the disease. The deviation between the standard values and the subject is used to establish the presence of the disease. As soon as the disease is established and a treatment protocol is initiated, hybridization assays can be repeated regularly to assess whether the level of expression in the patient begins to approximate that observed in the normal patient. The results obtained from successive trials can be used to demonstrate the effectiveness of the treatment during a period that varies from several days to months. The following are illustrations to illustrate the invention. They are not intended to limit the invention in any way. Example 1: Production and Analysis of VMCL-1 Cells The VMCL-1 cell line was made as follows. E14 rat mesencephalic cells, approximately 2-3 percent of which are glioblasts, were incubated in media containing 10 percent fetal bovine serum (volume / volume) for 12 hours and subsequently expanded in serum-free medium, which contained basic fibroblast growth factor (bFGF) as a mitogen. After more than 15 days in vitro, several islets of proliferation of glial-like cells were observed. After isolation and passage, the cells (referred to herein as VMCL-1 cells) proliferated rapidly either in serum-free medium or growth medium containing serum. The subsequent immunocytochemical analysis showed that it stained positive for two astrocyte markers, GFAP and vimentin, and negative for markers of oligodendro-glial or neuronal lineages, including A2B5, 04, GalC and MAP2. We have deposited the cell line VMCL-1 in the collection of American type cultures (Manassas, Virginia, United States, ATCC access No:, date of deposit, September 18, 2000). Serum-free conditioned medium, prepared from the VMCL-1 cell, caused increased survival and differentiation of E14 mesencephalic dopaminergic neurons in culture.
These actions are similar to those exerted by the conditioned medium derived from primary mesenteric astrocytes type-1. The expression of genes specific for mesencephalic regions (for example, wnt-1, en-1, en-2, pax-2, pax-5 and pax-8) was similar between VMCL-1 cells and type-type astrocytes. 1, primaries of ventral mesencephalic origin E14. In both, wnt-1 was strongly expressed, and at-1 less strongly supporting an expected expression factor of its mesencephalic origin. A chromosomal analysis showed that 70 percent of the cells were heteroploid, and of these 50 percent were tetraploid. No apparent decline in proliferative capacity was observed after more than 25 passages. The properties of this cell line are consistent with those of an immortalized type-1 astrocyte. VMCL-1 cells have a distinctly non-neuronal, glial-like morphology, but lack the large, flattened form typical of type-1 astrocytes in culture. The immunocytochemical analysis showed that they were stained positive for GFAP and vimentin, and negative for MAP2, A2B5 and 04. The cells therefore were not of the oligodendrocyte lineage. Based on the negative reaction to A2B5 and their morphological characteristics, they were not type-2 astrocytes either. The classification supported by immunocytochemical evidence is that of type-1 astrocytes, although, as noted, these cells lack the classical morphological features of primary type-1 astrocytes in culture. Example 2: Action of VMCL-1 CM in Dopaminergic Neurons E14 in Culture The CMVL-1 CM was tested at 0, 5, 20 and 50 volume / volume, to determine its ability to survive influence and development of mesencephalic dopaminergic neurons E14 of cultivation. The cultures were primed with 10 percent fetal bovine serum (FBS) for 12 hours, then cultured in serum-free culture medium thereafter, until stained and analyzed after 7 days in vitro. It was an action dependent on the dose of the conditioned medium on the increased survival of the dopaminergic neurons. The conditioned medium increased survival fivefold. In contrast, there was no significant increase in non-dopaminergic neuronal survival. The profile of the biological action of this putative factor is quite different from that of conditioned medium derived from glioma cell lines B49, the source of GDNF (Lin et al., Science 260: 1130-1132). Example 3: Gene Expression Analysis To further investigate the similarity between the VMCL-1 cell line and the primary cultured astrocytes, we measured the expression of six marker genes characteristic of the mesencephalic region. The analysis of wnt-1, en-1, en-2, pax-2, pax-5, and pax-8 showed that all genes were expressed in both the neural tissue of the ventral mesencephalon E13 and E14, with the exception of pax-2, which was expressed in E13 but not in neural E14. Both primary astrocytes and VMCL-1 cells expressed wnt-1 at levels comparable to ventral mesencephalic neural tissue E13 and E14. The expression side of en-1 was similar in primary astrocytes and VMCL-1 cells although at a lower level against expression in ventral mesencephalic tissue E13 and E14. In contrast, en-2, pax-5 and pax-8 were not expressed in primary astrocytes nor in VMCL-1. Pax-2 was expressed in E13 but not in ventral mesencephalon E14 and in primary astrocytes, but not in VMCL-1. Example 4: Chromosomal Analysis Chromosomes were counted in 34 cells. Of these, 9 had an account of 42, the diploid rat number. In the 25 cells that were heteroploid, 12/25 or 48 percent were in the tetraploid range. Hyperdiploid cells (counts of 43-48) hypodiploid (accounts of 39-41) each added 20 percent of the population, while 12 percent of extras had structurally rearranged chromosomes. The selective action of VMCL-1 CM to increase the survival of dopaminergic neurons in culture provides a potential clinical use for the molecule or molecules produced by this cell line. The lack of toxic action of. VMCL-1 CM at a concentration of 50 percent volume / volume indicates that the active neurotrophic factor, putative, it is not toxic. The action exerted by the VMCL-1 CM reflects almost exactly that of the conditioned medium prepared for mesencephalic astrocytes of the primary type-1 (Takeshima et al, J. Neurosci 14: 4769-4779, 1994). In high degree of putative factor specificity from VMCL-1 for dopaminergic neurons it is strongly indicated from the observation that general neuronal survival did not increase significantly, while the survival of dopaminergic neurons increased 5 fold. We have shown that primary type-1 astrocytes express GDNF mRNA, but no GDNF protein has been detected by Western staining in the conditioned medium at a sensitivity of 50 pg. Furthermore, we have shown that under the present experimental conditions, the increased survival of the dopaminergic neurons mediated by the optimal concentration of GDNF is never greater than 2 times. These observations alone indicate that the factor responsible for the neurotrophic actions of VMCL-1 CM is not GDNF. Example 5: Production of Conditioned Medium of Type-1 Astrocytes The conditioned medium of type-1 E16 astrocytes (10 liters) was filtered and applied to a column CL-6B sepharose with heparin (bed volume 80 ml) previously had been equilibrated with mM Tris-HCl (Mallinckrodt Chemical Co., Paris, Kentucky, United States) pH 7.6 containing 0.2 NaCl. After washing with equilibrium regulator, binding proteins of the column were eluted with a linear gradient of 0.2 M-2M NaCl in 20 mM Tris-HCl pH 7.6 (total volume 400 ml, flow rate 100 ml / hours). Fractions were collected using a LKB Pharmacia fraction collector and the absorbance was measured at 280 nm (Sargent-Welch PU 8600 UV / VIS Spectrophotometer). An aliquot of 1 ml was taken from each fraction, deposited in groups of four (total volume 4 ml) and desalted using Centricon-10® membrane concentrators (illipore, Bedford, Massachusetts, United States). The samples were diluted 1: 4 in defined medium and bioassayed to determine the dopaminergic activity. Active fractions were deposited (80 ml of total volume) then applied to a G-75 Sephadex® column (70 x 2.5 centimeters, Pharmacia Biotechnology Ltd, Cambridge, UK) which had been pre-equilibrated with 50 mM format Ammonium pH 7.4. The proteins were separated with the same regulator (flow rate 75 ml / hour) and the absorbance was measured at 280 nm. An aliquot of 1 ml was taken from each fraction, deposited in groups of four (total volume 4 ml), concentrated by lyophilization and reconstituted in 1 ml volume of distilled water. The samples were diluted 1: 4 in defined medium for dopaminergic bioassay. Those with neurotrophic activity were additionally bioassayed as individual fractions. An important distinguishing feature of VMCL-1 CM is that it predominantly promotes the survival of dopaminergic neurons, compared to the survival of neuronal GABAergic, serotonergic, and other neuronal phenotypes present in the culture. This specificity claim is also made for GDNF. The results of the extensive tests have shown, however, that the compound derived from VMCL-1 is not GDNF. In order to express the protein, an expression construct pcDNA3-hARP containing the cDNA of the human arginine-rich protein under the control of a CMV promoter was transiently transfected into COS cells and the conditioned medium was tested to determine the activity that promotes dopaminergic neuronal survival. A myc tag can be inserted to facilitate purification and immunodetection of the recombinant protein. Example 6: Isolation and purification of protein having dopaminergic neuronal test signal promotion activity The purification protocol was carried out as follows. All the salts used were of the highest purity and were obtained from Sigma Chemical Co. All the regulators were prepared recently and filtered through a 0.2 micron filter (vacuum driven Express GP system from Millipore). Step 1: Column chromatography of heparin-sepharose (4 ° C) Three liters of conditioned medium of VMCL-1 was diluted with an equal volume of 20 mM sodium phosphate buffer, pH 7.2 at room temperature, was filtered, and concentrated to a volume of 550 ml with a 0.226 m2 cartridge of 5K PREP / SCALE-TFF (Millipore). The concentrated material was loaded onto a 10 ml heparin-sepharose column assembled from 2 × 5 ml HiTrap heparin columns (Pharmacia Biotech) and pre-equilibrated with at least 100 ml of 10 mM phosphate buffer. sodium, pH 7.2 (regulator A). After the charge was complete, the column was washed with 100 ml of regulator A. A total of 10 fractions were eluted with regulator B (regulator A plus 1 M sodium chloride) in approximately volumes of 3 ml each. A sample of 300 μ? It was extracted for analysis. Step 2: Column chromatography of superose 12 (4 ° C) All fractions from step 1 were deposited, then concentrated to 4.5 ml using Centricon Plus-20 concentrator (5,000 MWCO, Millipore), loaded onto a filtration column in 16 x 600 mm gel packed with Superosa 12 medium (Prep Grade, Sigma Chemical Co.) and pre-equilibrated with at least 300 ml of 20 mM sodium phosphate buffer, pH 7.2 containing 0.6 M chloride of sodium chloride. sodium (GF regulator). Protein elution was carried out in GF regulator. Fractions of two ml were collected and analyzed for activity. The active protein was eluted in a volume of 15 ml after 84 ml of GF regulator was passed through the column and corresponded to approximately an elution region of 20-30 kiloDaltons based on column calibration data obtained with standards of protein (Bio-Rad). Step 3: Hydroxyapatite ceramic column chromatography (room temperature; FPLC system) The active fractions of step 2 that corresponded to the elution region of 20-30 kiloDaltons were deposited and concentrated to 7.5 ml, using a Centricon Plus-20 concentrator (5,000 MWCO), dialyzed overnight at 4 ° C against 2 liters of 10 mM sodium phosphate buffer, pH 7.2 (regulator A) and loaded (via Superloop) onto a 1 ml pre-packed ceramic hydroxyapatite column (Type I, Bio-Rad) equilibrated with regulator A After the excess of unbound protein (from the flow through) was washed from the column with regulator A, the linear gradient of regulator A containing 1.0 M NaCl was applied from 0 to 100 percent. Fractions of 1 millimeter were collected and analyzed to determine the activity. The active protein was eluted as a broad peak within the gradient regions corresponding to 0.4-0.8 M NaCl concentration. Step 4: Anion exchange column chromatography (room temperature, FPLC system) The fractions corresponding to the broad peak were deposited (total volume = 15 ml) and concentrated to 6 ml using Centricon Plus-20 (5,000 MWCO), dialyzed overnight at 4 ° C against 2 liters of 20 mM Tris HCl buffer, pH 7.5 (regulator A), was loaded (via Superloop) onto an anion exchange column of 1 ml FPLC (Uno, Bio-Rad), and equilibrated with regulator A. After the excess unbound protein was washed from the column with regulator A, a linear gradient of 0-100 percent of 1 M NaCl (in regulator A) was applied. Fractions of an MI were collected and analyzed to determine activity. The active protein was found in the flow through (ie, in the unbound protein fraction). Step 5: BioSil 125 column chromatography (room temperature, HPLC system) The active protein fraction of Step 4 (7 ml of total volume) was concentrated down to almost zero volume (approximately 1 μm using Centricon Plus-20 concentrator) (5,000 MWCO) and reconstituted in 0.6 ml of 10 mM sodium phosphate buffer, pH 7.2 The reconstituted material (70 L, analytical run) was loaded on a BioSil 125 HPLC gel filtration column (Bio-Rad ) equilibrated with 20 mM of sodium phosphate buffer, pH 7.2 (GF regulator) Chromatography was carried out using the HPLC system in HP 1100 series (Hewlett-Packard) .The levigado was collected in fractions of 120 μ? and it was analyzed to determine activity and protein content (SDS-PAGE) It was found that the activity in the fractions associated with the main absorbance peak of 280 nm was eluted from the column, which was represented by a protein of 45 kiloDaltons agree with the analysis by SDS-PAGE. However, no activity was found in the side fractions of the 45 kiloDalton protein peak, indicating that the activity could be due to the presence of another protein that was co-levigated with 45 kiloDaltons protein, but at a much lower concentration than it could be detected in the silver stained gel with 12 percent SDS-PAGE. Therefore, the remaining concentrated material from step 5 was further concentrated downward to a volume of 80 μ? using a Centricon-3 concentrator (Millipore), and 60 μ? It was commissioned and separated on the column under the same conditions as the analytical run described above. Aliquots of 8 μ? were taken from each fraction of 120 μ? of the levigated and analyzed by SDS-PAGE (12 percent gel) combined with silver staining. This analysis indicated that two additional proteins (which had molecular weights of approximately 18 and 20 kiloDaltons) were associated with active fractions and co-levigated with the protein greater than 45 kiloDaltons. The active fractions were dialyzed against 1 liter of ammonium acetate buffer, pH 8.0 (4 ° C) and combined to create two active deposits, Pl and P-2, so that Pl contained the protein of 20 kiloDaltons and the protein of 45 kiloDaltons, and P-2 contained the 18 kiloDaltons protein and the 45 kiloDaltons protein. Each deposit was dried on the SpeedVac vacuum concentrator (Savant) and reconstituted separately in 15 μ? of 0.1 ammonium acetate buffer, pH 6.9. The aliquots were removed from each sample and their activity was tested. Additionally, aliquots of 1 μ? they were subjected to 12% SDS-PAGE analysis followed by silver staining. The results of the above analyzes clearly indicated that P-1, but not P-2, contained the activity that promotes the desired survival. In the next step, both P-l and P-2 were dried over SpeedVac, reconstituted (each) in 10 μ? of freshly prepared SDS-PAGE reduction sample regulator (Bio-Rad), were incubated for one minute in a boiling water bath and loaded onto a 12 percent SDS-PAGE gel. After the electrophoresis was finished, the gel was fixed in a methane / acetic acid / water solution (50:10:40) for 40 minutes at room temperature, washed three times with nanopure water, and stained overnight with GelCode blue dye reagent (Pierce) at room temperature. After the inking was finished, and the GelCode solution was washed from the gene with nanopure water, the visible protein bands corresponding to the 45 kiloDaltons protein (both of Pl and P-2) and the 20 kiloDalton protein (only Pl) were separated from the gel with a razor. Each slice of gel containing a corresponding band was placed in a 1.5 ml microcentrifuge tube until the time of gel digestion. Example 7: Analysis of gel-digested fragments by nESI-MS / S The protein gel bands were incubated in 100 m of ammonium bicarbonate in 30 percent acetonitrile (aqueous) at room temperature for one hour in order to remove the colloidal Coomassie blue spot. The destaining solution was replaced several times until the dye was completely removed. The gel pieces were covered with deionized water (approximately 200 μm) and shaken for 10 minutes. The gel pieces were dehydrated in acetonitrile and, after removing the excess liquid, dried completely on a centrifuge evaporator. The gel bands were rehydrated with 20 μ? of 50 mM ammonium bicarbonate, pH 8.3, containing 200 ng of modified trypsin (Promega, Madison, Wisconsin, United States). The gel pieces were covered with 50 mM ammonium bicarbonate, pH 8.3 (approximately 50 μm), and incubated overnight at 37 ° C. The digested solutions were transferred to clean Eppendorf tubes and the gel pieces were sonicated for 30 minutes at 50-100 μ? of 5 percent acetic acid (aqueous). The extracted solutions were combined with digested solutions and evaporated to dryness on a centrifugal evaporator. The gel-digested extracts were first analyzed by mass spectrometry in flight at the time of matrix-assisted laser desorption (MALDI-TOFMS) using a Voyager Elite STR MALDI-TOFMS instrument (Applied Biosystems Inc., Framingham, assachusetts, United States) . The extracts were dissolved in 5 μ? of 50 percent acetonitrile, 1 percent acetic acid. Dihydroxybenzoic acid was used as the matrix and the spectra were acquired in positive ion, reflectron mode. Approximately one fifth of each sample was used for this analysis. These spectra provided the masses of the peptides in the digested extracts which were then used to search for a database of non-redundant protein sequences, at home, a process called fingerprints of peptide masses. The rest of the samples were used for peptide sequencing analysis by nanoelectro-dew ionization / tandem mass spectrometry (nESI -MS / MS). The extracts were first desalted using ZipTips C 18 (Millipo-re) and redissolved in 75 percent methanol (aqueous), 0.1 percent acetic acid (5 μ?). Approximately half of the samples were loaded in nanoelectro-dew glass capillaries (Micromass). NESI-MS / MS analysis was carried out using a four-pole Q-Star hybrid time-of-flight mass spectrometer (PE SCIEX, Concord, Ontario, Canada). All MS / MS analyzes were carried out in positive ion mode. The collision gas was nitrogen and the collision energy was 40-60 eV. The MS / MS spectra were typically acquired every second for a period of two minutes. The MS / MS spectra were used to search a database of non-redundant protein sequences at home using partial sequence tags (ie, only the peptide mass and some fragment ions were used for the database search If the protein was not identified by this procedure then the amino acid sequences of two or more peptides were determined as completely as possible from the MS / MS spectra These sequences were used to carry out BLAST searches on databases of protein sequences of NCBI, and EST sequences The results of the analysis identified the protein of 45 kiloDaltons in both Pl (active) and P-2 (inactive) and nexin derived from glia, and the protein of 20 kiloDaltons in Pl as protein rich in arginine, therefore, the protein rich in arginine is likely to be a major protein that is responsible for the activity observed in Pl, while the need for e the presence of nexin for the activity can not be excluded. Example 8: Generation of immortalized cell line for human mesencephalic tissue Using the methods described herein, a cell line derived from astrocyte type-1, having the same or similar activity that promotes neuronal survival, can be produced from of aborted human tissue. In humans, the corresponding gestational age of E14 is approximately 9-10 weeks, although other ages are also likely to be successful. The human compound is identified using standard protein purification techniques, as described herein. To induce a spontaneous immortalization of human fetal astrocytes, the ventral mesencephalic tissue is dissected from the human fetal brain. The dissection is preferably performed under sterile conditions in salt solution (eg, Hank's balanced salt solution (HABSS)), at pH 7.4.
The ventral mesencephalic (V) with the intact floor plate is localized, microdissected in a culture dish in fresh saline solution, completely cleared of non-neural tissue, and stored in salt solution. After the tissue collection, the salt solution is red, and the tissue is rinsed with two changes of culture medium (eg, N2), then dispersed in 2.0 ml of culture medium, which is used in all the subsequent procedures. The tissue is then crushed. Approximately 10 to 15 strokes are needed to completely disperse the cells. The cells are centrifuged (1,000 rpm, 2 minutes), the medium is aspirated, and the agglomerate dispersed in culture medium. The cells are counted using a hemocytometer, and the density is adjusted to approximately 2.5 x 10 5 cells / ml. The cells are then dispersed in culture dishes previously coated with polyornithine (15 mg / ml) and fibronectin (1.0 mg / ml), at a density of 5.0 x 10 4 cells / cm 2. The plates are transferred to the incubator (37 ° C, 5 percent C02, 100 percent humidity). Daily bFGF (10 nanograms / ml) is added, and the medium is changed every second day. At 8-12 days in vi tro, when the cultures have a confluence of approximately 50 percent, the cells are interrupted for 5 days. These conditions have previously shown that they cause a small percentage of the expanding astrocytes to spontaneously immortalize themselves. Alternatively, a cell line of human mesencephalic type-1 astrocytes can be established for primary cultures by transforming the cells with a DNA construct containing the early oncogenic region of SV40, under the control of transcription of the human GFAP promoter, and a selectable marker ( for example, pPGF-neo, which contains the murine phosphoglycerate kinase gene promoter). The transformants are selected with G418 and cloned. It has been previously shown that other transformed astrocytes retained characteristics consistent with the phenotype of type 1 astrocytes, including the immunoreactivity of GFAP and the high affinity mechanism of absorption for GABA that is inhibited by beta alanine (Radany et al., Proc. Nati, Acad. Sci. USA 89: 6467-6471, 1992). The previous results were obtained with the following methods. Mesencephalic Cultures A culture of primary mesencephalic cells was prepared from pregnant Sprague-Dawley rats (Taconic Farms; Germantown, New York, United States) as previously described (Shimoda et al., Brain Res. 586: 319-323, 1992; Takeshima et al, J. Neurosci 14: 4769-4779, 1994, Takeshima et al, Neuroscience 60: 809-823, 1994, Takeshima et al, J. Neurosci, Meth 67: 27-41, 1996). The dissected tissue was collected and deposited in oxygenated, cold HBSS (4 ° C), or medium containing 10 percent fetal bovine serum (Biofluids Laboratories, Rockville, Aryland, United States), depending on the purpose of the experiment. The pregnant rats were killed by exposing them to C02 on the fourteenth day of gestation (ie, E14), the abdominal region was cleaned with 70% EtOH, a laparotomy was performed, and the fetuses were collected and placed in saline. Regulated Dulbecco's phosphate (DPBS), pH 7.4, without Ca2 + or Mg +. The intact brain was red, a cut was made between the diencephalon and the mesencephalon, and the tectum was opened medially and extended laterally. The medial, ventral 1.0 mm3 tissue block comprising the mesencephalic dcpaminergic region was isolated. Blocks of tissue dissected were placed in cold oxygenated medium (4 ° C). The tissue was crushed without previous digestion. Alternatively, the cells were incubated in L-15 growth medium containing papain (Sigma Chemical Co), 10 U / ml, at 37 ° C, for 15 minutes, washed (3 x 2 ml) with a medium, and They mashed using only the needle and syringe. The dispersed cells were transferred to 1.5 ml Eppendorf tubes (1.0 ml / tube), and centrifuged at approximately 600 g for 2 minutes. The use of higher centrifugation rates over longer periods resulted in contamination of the cultures with residues and, as a result, sub-optimal growth of the cells. The medium was carefully aspirated, and the cells were resuspended in fresh medium and counted using a hemocytometer. All procedures, from laparotomy to plating, were completed within 2 hours. In a typical experiment, one liter of 10-15 fetuses produced 1.0 x 10 5 cells / fetus, or 1.0 x 106 - 1.5 x 10 6 cells / 1. Cultures of mesencephalic micro-islands To make cultures of mesencephalic micro-islands, the cells were prepared as described above, and resuspended at a final speed of 5.0 x 10 5 / ml. A drop of 25 μ? of the suspension (1.25 x 104 cells) was plated using a 100 μ pipette. on 8-well chamber slides with poly-D-lysine (50 μg / ml). The area of the drop was approximately 12.5 mm2, for a final average cell density of 1.0 x 105 / cm2. The drop was dosed uniformly, and the tip of the pipette was removed vertically, to avoid staining. The area occupied by the micro-island crop remained uniform for the duration of the crop. The cultures were incubated for 30 minutes at 37 ° C, at 5 percent C02 with 100 percent humidity, to allow the cells to bind, and 375 μl of culture medium was added to each well. The medium was changed after the first 12 hours, and approximately half of the medium was changed every second day after that. Cell Viability Assay A two-color fluorescence cell viability assay kit (viability / live / dead cytotoxicity assays, # L-3224, Molecular Probes, Inc., Eugene, Oregon, United States) it was used to identify living and dead cells before plating and in cultures. Living and dead cells fluoresce green and red, respectively, giving two positive indicators of viability. The ethidium homodimer and calcein-AM were diluted with DPBS to give final concentrations of 3.8 μ? and 2.0 μ ?, respectively. The evaluation of the viability of the cells was made before plating. A cell suspension was incubated for 15 minutes with an equal volume of dye (typically 20 / iL) on glass slides at room temperature in a dark, moist chamber with a sliding cover, and then examined with a fluorescent microscope using FITC optics. . The viability of the cells just before plating was approximately 95 percent. Culture Medium The serum-free medium used consisted of equal volumes of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (Gibco, Grand Island, New York, United States).; 320-1320AJ), 1.0 mg / ml bovine albumin fraction V (Sigma Chemical Co., A-4161), 0.1 μg / ml apo-trans-errine (Sigma, T-7786), 5 μg / ml insulin (Sigma; 1-1882), 30 nM of L-thyroxine (Sigma, T-0397), 20 nM progesterone (Sigma, P-6149), 30 nM of sodium selenite (Sigma, S-5261), 4.5 mM glutamine (Gibco, 320-5039AF), 100 U / mL of penicillin, and 100 and g / mL of streptomycin (Gibco, P-100-1-91). Preparation of conditioned medium for the VMCL-1 cell line To prepare conditioned medium for the VMCL-1 cell line, 2.0 x 10 6 cells were plated in a 15-centimeter uncovered culture dish in 20 ml of culture medium containing 1.0 percent of fetal calf serum. At 80 percent confluence, the medium was aspirated and the cells were washed once with serum-free medium. 20 ml of serum free N2 medium without albumin was added, and the conditioning allowed to continue for 48 hours. During this time, the cells also expanded to a 100 percent confluence. The medium was aspirated, placed in 50 ml tubes, centrifuged (15,000 rpm for 20 minutes) and then placed in a 1.0 liter plastic bottle. Usually 5 ml of each batch of conditioned medium was filtered sterilized using a 0.22 micron filter, stored in 5 ml aliquots, at a temperature of -70 ° C, and used to determine neurotrophic potency, before being deposited with a larger warehouse of conditioned medium. If desired, conditioned medium of VMCL-1 can be made in large quantities using standard industrial cell culture techniques known to those skilled in the art. Production of conditioned medium for type-1 astrocytes Type 1 astrocytes were prepared as follows. Rat fetal brain stem E16 was dissected in cold DPBS, and the mesencephalic region was transferred to astrocyte culture medium (DMEM / Ham F-12, 1: 1, 15 percent FBS, 4.0 mM glutamine, 30 nM selenite sodium, penicillin, and streptomycin). Cells were dispersed by trituration in 2 ml of fresh medium using an 18-gauge needle fitted to a syringe. The cells were centrifuged for 5 minutes at 2,000 rpm in a centrifuge, resuspended in medium and ground again. The final cell agglomerate was dispersed and plated at a density of 1 x 10 6 cells / 75 cm 2 of flask in 15 ml of medium. The cells were incubated at 37 ° C in a 5 percent atmosphere of carbon dioxide and 95 percent air for 24 hours, and the cells that did not join were removed by aspiration. The cells were cultured for an additional nine days, and the flasks were shaken vigorously for 16 hours to remove any contaminating cell type. The monolayers of astrocytes were washed three times with DPBS, trypsinized and replated (density of 1 x 106 cells / flask). At this time, a small portion of cells were plated on eight-well chamber slides (Nunc Inc., Naperville, Illinois, United States); these sister cultures were treated as described for flask cultures. At the confluence, the medium was replaced with media containing 7.5 percent FBS and the cells were incubated for 48 hours. In the following exchange, defined serum free medium (DMEM / Ham F-12, 1: 1, 4.0 mM glutamine, 30 nM sodium selenite, 100 U / ml penicillin and 100 mg / ml streptomycin) was added and the cells were incubated for another 48 hours. The medium was replaced and, after five days, the conditioned medium was harvested and mixed with leupeptin (lOmM: Bachem, Torrance, California, United States) and 4- (2-aminoethyl) -benzenesulfonyl fluoride hydrochloride (1.0 mM). : ICN Biochemicals, Aurora, Ohio, United States) to inhibit proteolysis. At the time of harvest, the monolayers of astrocytes grown on chamber slides were immunostained in order to assess the phenotype of the culture. VMCL-1 cell culture To grow VMCL-1 and prepare VMCL-1 CM, 2.0 x 106 cells were plated in a 15 cm uncovered culture dish in 20 ml of culture medium that initially contained 10 percent fetal bovine serum. At an 80 percent confluence, the medium was aspirated and the cells were washed once with serum-free medium. Usually 20 ml of serum free medium without albumin was added, and the conditioning was allowed to continue for 48 hours. The N2 medium proved to be particularly convenient for use to collect the conditioned medium. During these 48 hours, the cells usually expanded to a 100 percent confluence. The medium was aspirated, placed in 50 ml tubes, centrifuged (15,000 rpm, 20 minutes) and placed in a one liter plastic bottle. Approximately 5 ml of each batch of conditioned medium was sterilized using a 0.22 mm filter, stored at 0.5 ml aliquots, at -70 ° C, and used to determine neurotrophic potency before being deposited with the largest storage. of conditioned medium. The cell line V CL-1 had now passed more than 50 times. Immunocytochemistry For TH and MAP2 immunocytochemistry, the cultures were washed (2 x 250 μ) with cold DPBS, fixed with 4 percent folmaldehyde in phosphate buffered saline for 10 minutes, permeabilized using 1 percent CH3COOH / 95 percent EtOH at -20 ° C, for 5 minutes, and then washed (3 x 250 μ?) With phosphate buffered saline. The non-specific binding was blocked with 1 percent bovine serum albumin in phosphate buffered saline (BSA-PBS) for 15 minutes. Anti-TH anti-body (50 μ?) (Boehringer-Mannheim, Indianapolis, Indiana, USA), or anti-MAP2 anti-body (Boehringer-Mannheim) was applied to each well, and the slides were incubated in a box wet dark at room temperature for 2 hours. Control staining was done using mouse serum at the same dilution as the anti-TH anti-body. After washing (2 x 250 μ?) With phosphate buffered saline, anti-mouse IgG-FITC (50 μ?) Was applied, and the slides were incubated for an additional 1 hour. After washing with phosphate buffered saline (2 x 250 μ), the excess fluid was aspirated, the walls of the chamber were removed, and a single drop of Vecta Shield mounting medium was applied (Vector Laboratories, Burlingame, California, United States), followed by a cover glass, which was sealed with nail varnish.
In some experiments, TH was identified using biotinylated secondary anti-bodies, and the product of the diaminobenzidine (DAB) reaction enhanced with nickel was developed using the biotinylated peroxidase-avidin complex (ABC kit, Vector Laboratories). For the fibrillar acid glial protein (GFAP, Boehringer-Mannheim, # 814369), fixation and permeabilization were made in one step using 5 percent CH3COOH / 95 percent C2H5OH at -20 ° C. Subsequent procedures were the same as those used to visualize the TH. For A2B5 and 04, the cultures were washed with cold DPBS (2 x 250 μ?) And blocked with 1 percent BSA-PBS for 10 minutes. The anti-body A2B5 (50 μ?) Was applied to each well, and incubated for 1 hour. After washing with DPBS (2 x 250 μ?), The secondary anti-body, anti-IgM-FITC, was applied for 30 minutes. The cells were then washed with DPBS (2 x 250 μ?). To counterstain the cell nuclei, the cells were incubated with 0.5 g / ml nucleic acid dye H33258 (Hoechst, Kansas City, Missouri, United States) in 10 mM sodium bicarbonate for 15 minutes at room temperature, then rinsed in Saline regulated phosphate solution for 2 x 10 minutes. After the final wash with cold DPBS (2 x 250 μ), they were mounted as described above. RT-PCR analysis Total RNA was extracted from rat ventral mesencephal tissue E13 or E14 or from 1 x 109 astrocytes or from 1 x 109 VMCL-1 cells using RNA-STAT reagent (TelTest, University of Maryland, Baltimore, Maryland, U.S) . The first strand cDNA was generated from the RNA and amplified by polymerase chain reaction using the manufacturer's procedures. The products of the reaction were resolved by 2 percent agarose gel electrophoresis to determine the size and relative abundance of the fragments. The results of the polymerase chain reaction for β-actin and GAPDH were included as controls to confirm the equal loading of cDNA. Chromosomal analysis The cells were cultured in DMEM / F-12 1: 1 supplemented with 2.5 percent fetal bovine serum, D-glucose (2.5 g / 1) and -ITS supplement, diluted 1: 100. Twenty-four hours later, subcultures in the metaphase stage were stopped with colchicine (10 ^ g / ml). The cells were trypsinized and subjected to hypotonic shock (75 mM KC1). The cells were then fixed in three changes of MeOH / CH3COOH, 3: 1, and dried in air. The cells were stained using 4 percent Geisma, and examined microscopically. Deposit The applicant made a deposit of at least 25 vials containing the VMCL-1 cell line in the American-type crop collection, Manassas Virginia, 20110 United States
United, ATCC deposit number. The cells were deposited with the ATCC on September 18, 2000. This VMCL-1 deposit will be kept in the ATCC deposit, which is a public deposit, for a period of 30 years, or 5 years after the most recent request. , or during the effective life of the patent, and if it becomes longer, it will be replaced if it becomes non-viable during that period. Additionally, the applicant has satisfied all the requirements of 37 C.F.R. ??? .801-1.809, including an indication of the viability of the sample. The applicant does not impose restrictions on the availability of the deposited material of the ATCC. The applicant does not have the authority, however, to waive the restrictions imposed by law on the transfer of biological material or its transportation in commerce. The applicant does not waive any infringement of its rights granted under this patent. Other Forms of Realization All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to the preferred specific embodiments, it should be understood that the invention as claimed should not be limited to these specific modalities. However, it is intended that various modifications of the modes described to carry out the invention, which are obvious to those skilled in the field of molecular biology or related fields, are within the scope of the invention.
LIST OF SEQUENCES < 110 > Neurotrophic Bioscience Inc. < 120 > Promoter Factors of Neuronal Survival, Dopaminergi-cos, and their uses
< 210 > 1 < 211 > 179 < 212 > PRT < 213 > Homo Sapiens < 400 > 1 Met Trp Wing Thr Gln Gly Leu Wing Val Arg Val Wing Leu Ser Val Leu
1 5 10 15
Pro Gly Ser Arg Ala Leu Arg Pro Gly Asp Cys Glu Val Cys lie
20 25 30 Tyr Leu Gly Arg Phe Tyr Gln Asp Leu Lys Asp Arg Asp Val Thr Phe
35 40 45 Ser Pro Ala Thr lie Glu Asn Glu Leu lie Lys Phe Cys Arg Glu Ala
50 55 60 Arg Gly Lys Glu Asn Arg Leu Cys Tyr Tyr lie Gly Ala Thr Asp Asp
65 70 75 80
Ala Ala Thr Lys lie lie Asn Glu Val Ser Lys Pro Leu Ala His His 85 90 95 lie Pro Val Glu Lys lie Cys Glu Lys Leu Lys Lys Lys Asp Ser Gln
100 105 110 lie Cys Glu Leu Lys Tyr Asp Lys Gln lie Asp Leu Ser Thr Val Asp
115 120 125 Leu Lys Lys Leu Arg Val Lys Glu Leu Lys Lys lie Leu Asp Asp Trp
130 135 140 Gly Glu Thr Cys Lys Gly Cys Wing Glu Lys Ser Asp Tyr lie Arg Lys
145 150 155 160 lie Asn Glu Leu Met Pro Lys Tyr Ala Pro Lys Ala Wing Ser Wing Pro 165 170 175
Thr Asp Leu < 210 > 2 < 211 > 179 < 212 > PRT < 213 > Mus musculus < 400 > 2 Met Trp Wing Thr Arg Gly Leu Wing Val Wing Leu Wing Leu Ser Val Leu
1 5 10 15 Pro Asp Ser Arg Ala Leu Arg Pro Gly Asp Cys Glu Val Cys lie
20 25 30 Tyr Leu Gly Arg Phe Tyr Gln Asp Leu Lys Asp Arg Asp Val Thr Phe
35 40 45 Ser Pro Ala Thr lie Glu Glu Glu Leu lie Lys Phe Cys Arg Glu Ala
50 55 60 Arg Gly Lys Glu Asn Arg Leu Cys Tyr Tyr lie Gly Ala Thr Asp Asp
65 70 75 80
Ala Ala Thr Lys lie lie Asn Glu Val Ser Lys Pro Leu Ala His His 85 90 95 lie Pro Val Glu Lys lie Cys Glu Lys Leu Lys Lys Lys Asp Ser Gln
100 105 110 lie Cys Glu Leu Lys Tyr Asp Lys Gln lie Asp Leu Ser Thr Val Asp
115 120 125 Leu Lys Lys Leu Arg Val Lys Glu Leu Lys Lys lie Leu Asp Asp Trp
130 135 140 Gly Glu Met Cys Lys Gly Cys Wing Glu Lys Ser Asp Tyr lie Arg Lys
145 150 155 160 lie Asn Glu Leu Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ala Arg 165 170 175
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< 210 > 4 < 211 > 9 < 212 > PRT < 213 > Rattus norvegicus < 400 > 4 Gln lie Asp Leu Ser Thr Val Asp Leu 1 5
< 210 > 5 < 211 > 1103 < 212 > DNA < 213 > Homo sapiens < 400 > 5 cttcggtcct gctgtagtgc cttctgcgcc aggcccggtt caatcagcgg ccacaactgt 60 gacaccacca ctagggctca gccaatgagg gagggcacgt ggagccgcgt ctgggctcgc 120 ggctcctgac caatggggaa gtggcatgtg ggagggcgcc ggggttcccc ccgccaatgg 180 ggagctacgg cgcgcggccg ggacttggag gcggtgcggc gcggcgggtg cggttcagtc 240 ggtcggcggc ggcagcggag gaggaggagg aggaggagga tgaggaggat gaggaggatg 300 tgggccacgc aggggctggc ggtgcgcgtg gctctgagcg tgctgccggg cagccgggcg 360 ctgcggccgg gcgactgcga agtttgtatt tcttatctgg gaagatttta ccaggacctc 420 aaagacagag atgtcacatt ctcaccagcc actattgaaa acgaacttat aaagttctgc 480 cgggaagcaa gaggcaaaga gaatcggttg tgctactata tcggggccac agatgatgca 540 tcatcaatga gccaccaaaa ggtatcaaag cctctggccc accacatccc tgtggagaag 600 agcttaagaa atctgtgaga gaaggacagc cagatatgtg agcttaagta tgacaagcag 660 gcacagtgga atcgacctga cctgaagaag ctccgagtta aagagctgaa gaagattctg 720 gatgactggg gggagacatg caaaggctgt gcagaaaagt ctgactacat ccggaagata 780 tgcctaaata aatgaactga tgcccccaag gcagccagtg caccgaccga tttgtagtct 840 gctcaatctc tgttgcac ct gagggggaaa aaacagttca actgcttact cccaaaacag 900 cctttttgta atttattttt taagtgggct cctgacaata ctgtatcaga tgtgaagcct 960 ggagctttcc tgatgatgct ggccctacag tacccccatg aggggattcc cttccttctg 1020 ttgctggtgt actctaggac ttcaaagtgt gtctgggatt tttttattaa agaaaaaaaa 1080 tcaaaaaaaa tttctagctg aaa 1103