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Immunological detection of prostate diseases and prostasome-related conditions |
The present invention relates to a diagnostic and/or prognostic reagent comprising a component which selectively binds to anti-prostasome autoantibodies, as well as an immunoassay using said reagent. Furthermore, the invention relates to an in vitro method for diagnosing and/or prognosticating a condition reflecting the prostasome presence in body fluids, comprising a) binding of anti-prostasome autoantibodies with a component which selectively binds to anti-prostasome autoantibodies; and b) detection of said binding. The conditions may be different forms of prostate cancer or prostasome-related diseases or other conditions where anti-prostasome autoantibodies are present in the body. |
1. An agent which selectively binds to anti-prostasome auto antibodies. 2. An agent according to claim 1, which is a diagnostic and/or prognostic reagent. 3. An agent according to claim 1, comprising structures involved in the binding of prostasomes or prostasomal-like material and anti-prostasome autoantibodies. 4. A kit for diagnosis/prognosis of prostasome-related diseases comprising a reagent according to claim 1. 5. An immunoassay for detection of anti-prostasome autoantibodies in body fluids comprising the reagent according to claim 2. 6. An immunoassay according to claim 5, wherein the body fluid is serum or plasma. 7. An immunoassay according to claim 5 wherein the agent is bound to a solid support. 8. An immunoassay according to claim 5, wherein the solid support is a microtitre plate. 9. An immunoassay according to claim 5 which is an ELISA or flow cytometry. 10. An in vitro method for diagnosing and/or prognosticating a condition reflecting the prostasome presence in body fluids, comprising: a) binding of anti-prostasome autoantibodies with a component which selectively binds to anti-prostasome autoantibodies; and b) detection of said binding. 11. A method according to claim 10, wherein said component in a) comprises structures involved in the binding of prostasomes or prostasomal-like material and anti-prostasome autoantibodies. 12. A method according to claim 10, wherein the condition is cancer. 13. A method according to claim 10, wherein the condition is prostasome-related disease. 14. A method according to claim 10, wherein the condition is prostate cancer. 15. A method according to claim 10, wherein the condition is prostatic disease. 16. A method according to claim 10, wherein the detection in step b) is by ELISA or flow cytometry techniques. 17. (canceled) 18. A method according to claim 11, wherein the condition is cancer. 19. A method according to claim 11, wherein the condition is prostasome-related disease. 20. A method according to claim 11, wherein the condition is prostate cancer. 21. A method according to claim 11, wherein the condition is prostatic disease. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Prostate cancer is the fourth most commonly diagnosed cancer in men worldwide and the most commonly diagnosed in Swedish men. The age-adjusted incidence of prostate cancer has increased by about 1.8% per year over the past decade. This increase may be partly due to the introduction of better diagnostic and therapeutic techniques. The prevalence of prostate cancer is comparatively low in men younger than 60 years in Sweden but is 4% in men 75-79 years of age and is as high as 5-6% in men over the age of 80 years. Autopsy studies have indicated a much higher prevalence of latent prostate cancer. Hence, a prevalence of 44% was found in men 50-59 years of age and 83% in those 70-79 years, which suggests that many prostate cancers do not reach a clinically significant stage. In Sweden, 6,610 new cases of prostate cancer were reported in 1998; 67% of these men were older than 70 years, and only 0.26% were younger than 50 years. In 1997, 2,448 men died of prostate cancer indicating substantial mortality from this cancer form. It has also been shown that the younger the patient is at the time of diagnosis, the higher is the mortality. The causes of prostate cancer are essentially unknown, although several factors have been shown to be associated with a higher risk for this type of cancer as increasing age, family history of prostate cancer, men in Western countries, and especially American men of African heritage. Nonbacterial prostatitis is a benign and common disease mostly among young men. The etiology of the disease is not fully understood. The symptoms include genital and pelvic pain, urgency to void and cold sensitivity. Although the symptoms are suggestive of an infection in the prostate gland, there is a lack of clear bacterial infectious etiology for a majority of men with these symptoms. Among alternative etiologic factors, it has been claimed that an autoimmune component to the disease might exist. This means that a self-reactivity directed against the prostate or a prostate component by the immune system could be involved in the etiology of the disease. Accordingly, a production of autoantibodies against some component of the prostatic secretion in patients with nonbacterial prostatitis could contribute to their symptoms. Autoantibodies can be produced in the body and cause severe diseases, like rheumatic artritis and a type of diabetes, by attacking cells carrying the corresponding antigens. No autoantibodies, which recognize or affect cancer metastases, have yet been reported. The prostate gland is one of the major accessory genital glands with mainly exocrine functions. Although being a unified organ, three pairs of lobes can be distinguished. Histologically the prostate is composed of a large series of independent branching ducts, all of which enter the prostatic urethra. Hence, the prostate gland and its secretion represent a closed secretory system and the secretory components will normally not be able to appear in the circulation. Human prostatic fluid contains high amounts of monovalent and divalent cations. It is also rich in enzymes involved in carbohydrate metabolism and protein degradation. Besides these soluble substances the prostate gland secretes the above-mentioned prostasomes which are prostatic secretory products coating the sperm cells. Prostasomes are surrounded by a bilayered and sometimes tri- or multi-layered membrane and they have a diameter of 40-500 nm. Prostasomes are secretory products of the prostate gland. The membrane architecture of these organelles is complex and two-dimensional gel electrophoresis of membrane material has revealed about 80 different protein entities. Also, an unusually high cholesterol/phospholipid ratio is inherent in this membrane. The prostasomes contain neuroendocrine and CD molecules and many different enzymes are part of the prostasome membrane mosaic. Prostasomes have been ascribed many different biologic activities, but their physiologic function is still unclear. They can interact with spermatozoa and promote their motility characteristics in different ways. They are also immunosuppressive and inhibit superoxide anion generation by neutrophil granulocytes. The prostasomes can modulate complement-mediated immune responses, and CD 59, an inhibitor of the membrane attack complex of complement, resides on prostasomes. The present inventors have previously isolated and purified prostasomes not only from seminal plasma and expressed prostatic fluid (exprimate) but also from the human prostate gland as well as from vertebrate metastases of prostatic cancer. They have also cultured human prostatic cancer cells of the PC3 and similar cell lines on plates in monolayer and found that they can produce prostasomes. These PC3 cells have been fractionated and the prostasomes purified C3 prostasomes). In addition, the present inventors and others have produced monoclonal (1, 3-4) and polyclonal (2) antibodies against some types of prostasomes. Historically, prostate cancer was most often diagnosed in men presenting with symptoms derived from a local tumour or metastatic spread of a tumour, such as dysfunctional voiding or bone pain, and the disease was at an advanced stage at the time of diagnosis. Occasionally, it was an accidental finding on digital rectal examination or upon histological examination of tissue obtained during surgery on men with benign prostatic hyperplasia. Accordingly, there is a need of improvement and intensified use of diagnostic procedures on men at risk, resulting in more extensive detection of nonlethal prostate cancers. Measurement of prostate specific antigen (PSA) in serum was initiated in the latter half of the 1980s in many Western countries. This measure changed the pattern of diagnosis of prostate cancer with more cases detected at an early stage and fewer cases at advanced stages. However, since PSA is not a prostate cancer specific marker in serum it is not the ideal diagnostic marker and therefore not accommodated for screening of prostate cancer. There is growing concern about the PSA-test, as it is employed at present. The test is hampered by its incapacity for discriminating in a proper way between benign prostatic hyperplasia and prostate cancer and, what is more, between prostate cancer with high metastasising potential (aggressive prostate cancer) and such cancer with no or weak aggressiveness. This inability of the test often will end up in truncating surgery, i.e. total prostatectomy. This type of overtreatment is a great inconvenience not only for patients but also for society due to extra costs. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based upon the demonstration of anti-prostasome autoantibodies in serum of patients with prostate cancer due to the border-breaking growth of neoplastic cells in the prostate. The invention also comprises the idea that prostasomes, due to their smallness, will appear in the circulation earlier than the much bigger (about 150 times) prostate cancer cells, which are spread via the blood. Accordingly, a time-window may be offered for the prostasomes, during which the anti-prostasome autoantibodies can be developed and detected by our test, before the bigger prostate cancer cells will be released into circulation and set their metastases. The anti-prostasome autoantibodies in body fluids (here represented by blood plasma/serum) may, for example, be detected with an ELISA technique (see below). Herewith, the invention presents a new way for diagnosing early metastasis and for discriminating between the dangerous (metastasis-prone) prostate cancer and the more or less harmless (not metastasis-prone) variant of prostate cancer. The anti-prostasome autoantibodies, which we have detected in serum from patients with a prostate cancer, do not attack the cancer cells and are therefore not recognized clinically. In a first aspect, the invention relates to an agent which selectively binds to anti-prostasome autoantibodies. In one embodiment, the agent is a diagnostic and/or prognostic reagent comprising a component which selectively binds to anti-prostasome autoantibodies. The component should comprise structures involved in the binding of prostasomes and anti-prostasome autoantibodies, for example prostasomes or epitope(s) exposed in prostasomes binding to anti-prostasome antibodies, i.e. with selective affinity to anti-prostasome autoantibodies. By the term ‘selective’ is also meant that the binding is mainly selective. The reagent (i.e. antigen) may comprise prostasomal-like material, for example, of natural, pathological, synthetic or recombinant origin. In a second aspect, the invention relates to a kit for diagnosis/prognosis of prostasome-related diseases comprising a reagent as defined above. In a third aspect, the invention relates to an immunoassay for detection of anti-prostasome autoantibodies in body fluids comprising the above reagent. The body fluid may be serum or plasma. Further alternatives are urine and semen. Optionally, the reagent (i.e. the antigen) is bound to a solid support, such as a microtitre plate which is commonly used in, for example, ELISA. The immunoassay according to the invention may be combined with a conventional PSA assay if desired. In a fourth aspect, the invention relates to an in vitro method for diagnosing and/or prognosticating a condition reflecting the prostasome presence in body fluids, comprising a) binding of anti-prostasome autoantibodies with a component which selectively binds to anti-prostasome autoantibodies; and b) detection of said binding. Preferably, the detection is by by ELISA or flow cytometry techniques. The condition may be cancer, such as prostate cancer, or prostatic diseases. For instance, the demonstration of anti-prostasome autoantibodies in body fluids may be important to better differentiate between bacterial and nonbacterial prostatitis (see above). An improved differential diagnosis between these two conditions would facilitate the position that should be taken to the question of proper treatment. In a fifth aspect, the invention relates to use, or method of using, of an agent which selectively binds to anti-prostasome autoantibodies for the production of a drug for prevention and/or treatment of prostasome-related diseases. For example, the drug may be formulated as a vaccine. detailed-description description="Detailed Description" end="lead"? |
Methods of determining west nile virus epitopes and method of using the same |
Vaccines containing one or more West Nile Virus (WNV) vaccine candidate peptides in an immunologically acceptable excipient are disclosed. Also provided are recombinant WNV vaccine candidate peptides, wherein the peptide is expressed from a recombinant polynucleotide such as a naked DNA vaccine. Additionally, methods for inducing anti-WNV immune responses in a mammalian subject are also disclosed. |
1. A vaccine comprising: one or more West Nile Virus (WNV) vaccine candidate peptides selected from the group consisting of SEQ ID NO: 1-95, in an immunologically acceptable excipient. 2. The vaccine of claim 1, wherein the peptide is between 8 amino acids and 10 amino acids in length. 3. The vaccine of claim 1, wherein one or more of the WNV vaccine candidate peptides has an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20. 4. The vaccine of claim 1, wherein the peptide is complexed to a carrier protein. 5. The vaccine of claim 1, wherein the peptide is a recombinant fusion protein. 6. The vaccine of claim 1, wherein the excipient is an adjuvant. 7. A recombinant WNV vaccine candidate peptide, comprising: a peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20, wherein the peptide is expressed from a recombinant polynucleotide. 8. The recombinant peptide of claim 7, wherein the recombinant polynucleotide is a naked DNA vaccine. 9. A method for inducing an anti-WNV immune response, comprising: administering to a mammalian subject the vaccine according to claim 1. 10. The method of claim 9, wherein the induction of an anti-WNV immune response results in the raising of an anti-WNV antibody. 11. The method of claim 9, wherein the mammalian subject is a human. 12. The method of claim 9, wherein the vaccine is administered orally, topically, parenterally, by viral infection, or intravascularly. 13. A method for inducing an anti-WNV immune response, comprising administering to a mammalian subject the vaccine candidate peptide according to claim 7. 14. The method of claim 13, wherein the induction of an anti-WNV immune response is the raising of an anti-WNV antibody. 15. The method of claim 13, wherein the mammalian subject is a human. 16. The method of claim 13, wherein the vaccine is administered orally, topically, parenterally, by viral infection, and intravascularly. |
<SOH> BACKGROUND <EOH>West Nile virus (WNV) is the cause of a potentially fatal form of viral encephalitis that suddenly emerged in the New York City area during 1999. The virus is a member of the flavivirus family. Other members of the same family include St. Louis Encephalitis, Japanese Encephalitis Virus (JEV), Hepatitis C and Dengue. WNV is commonly found in West Asia, Africa, and the Middle East but was not reported in the Americas until 1999. (Lanciotti et al., Science 286:2333-37 (1999); Wright et al., Aust. J. Exp. Biol. Med. Scie. 61(Pt. 6):641-53 (1983)). The source of the introduction of the virus to New York City is unknown. Introduction by an infected host (e.g. human or bird), by an infected vector (e.g. mosquito), or by bio-terrorists are potential sources of WNV listed by the United States Centers for Disease Control. Surveillance data reported to the CDC have indicated intensified transmission and geographic expansion of the West Nile Virus (NY99) outbreak in the northeastern United States during the last two years. Twelve states and the District of Columbia reported WNV epizootic activity in 2000, a significant increase over the four states reporting activity in 1999. West Nile Virus is expected to continue to spread along the East Coast of the United States in 2001 and years thereafter due to over-wintering of mosquitoes and avian migratory patterns. (Andersen et al., Science 286:2331-33 (1999); Rappole et al., Emerging Infectious Diseases 6(4):319-28 (2000)). Concern about the dissemination of WNV in the United States is supported by knowledge of current endemics and epidemics in other regions of the world. The largest African epidemic, with approximately 3,000 clinical cases, occurred in South Africa after heavy rains in 1974. Other outbreaks have been observed in the former Soviet Republic, Central African Republic, Kisangani in the Democratic Republic of Congo (former Zaire), Egypt, Ethiopia, India, Israel, Madagascar, Nigeria, Pakistan, Senegal, Sudan, and quite a few European countries. The West Nile NY99 virus that was eventually associated with the New York 1999 outbreak appears to have been circulating in Israel since 1997. Other close relatives to the West Nile NY99 virus were isolated in Italy (1998), Morocco (1996), Romania (1996), and Africa (1989, 1993, 1998). The epitope-driven vaccine concept is an attractive one that is being successfully pursued in a number of laboratories. See, e.g., Hanke et al., Vaccine 16:426 (1998); Ling-Ling et al., J. Virology 71:2292-302 (1997); Nardin et al., Immunol. 166(1):481-89 (2001). |
<SOH> SUMMARY OF THE INVENTION <EOH>The goal of this project was to demonstrate the utility of a bioinformatics/computational immunology approach for the rapid selection of epitope reagents that would permit the evaluation of cell-mediated responses in the immunopathogenesis of West Nile Virus (WNV). In one embodiment, the invention is concerned with the development of diagnostic reagents such as tetramers and preventive or therapeutic vaccines. (Altman et al., Science 274(94):6 (1996)). In one aspect, the invention includes a vaccine that includes one or more West Nile Virus (WNV) candidate peptides disclosed in SEQ ID NOs: 1-95 and is in an immunologically acceptable excipient. For example, the vaccine could contain a combination of 2, 3,4, 5, 6, etc., WNV peptides disclosed in SEQ ID NOs:1-95. In one embodiment, the invention includes a vaccine where the length of one or more WNV candidate peptides is between 8 amino acids and 10 amino acids in length. In another embodiment, the invention includes a vaccine where one or more WNV candidate peptides have amino acid sequences from the group disclosed in SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20. The invention also includes a vaccine containing one or more WNV candidate peptides, where the peptide is complexed to a carrier protein. The carrier protein may be a recombinant fusion protein. Additionally, the excipient may be an adjuvant. In another aspect, the invention includes one or more recombinant WNV candidate peptide where the peptides contain an amino acid sequence from the group disclosed in SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20 and is expressed from a recombinant polynucleotide. In one embodiment, the recombinant polynucleotide is a naked DNA vaccine. In another embodiment, the invention involves a method for inducing an anti-WNV immune response by administering a vaccine containing one or more WNV vaccine candidate peptides selected from the group consisting of SEQ ID NOs:1-95 and an immunologically acceptable excipient to a mammal. In a further embodiment, the induction of an anti-WNV immune response results in the raising of an anti-WNV antibody. In various embodiments, suitable mammals include, for example, humans, cows, pigs, horses, and dogs. Administration of the vaccine according to the invention may be orally, topically, parenterally, by viral infection, and/or intravascularly. In another embodiment, the invention involves a method for inducing an anti-WNV immune response by administering a vaccine candidate peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 8, 9, 13, 15, and 17-20, wherein the peptides are expressed from a recombinant polynucleotide. In a further embodiment, the induction of an anti-WNV immune response results in the raising of an anti-WNV antibody. In various embodiments, suitable mammals include, for example, humans, cows, pigs, horses, and dogs. Administration of the vaccine according to the invention may be orally, topically, parenterally, by viral infection, and/or intravascularly. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and claims. |
Cloning of transgenic animals comprising artificial chromosomes |
The invention is directed in part to totipotent cells that have one or more artificial chromosomes; processes for producing such cells; processes for using such cells (e.g., nuclear transfer); transgenic embryos and transgenic animals cloned from such cells; and processes for producing such embryos and animals. |
1. A method of producing a transgenic ungulate embryo by nuclear transfer of a nuclear donor cell into an enucleated recipient cell, the method comprising: (a) fusing a nuclear donor cell, or a nucleus thereof, with an enucleated recipient cell of the same species as the nuclear donor cell to form a nuclear transfer embryo, wherein a heterologous DNA molecule of greater than 100 kilobase pairs is injected into said oocyte prior to or following fusion, whereby said nuclear transfer embryo comprises said heterologous DNA molecule; and (b) activating the nuclear transfer embryo to provide said transgenic ungulate embryo. 2. A method according to claim 1, wherein said transgenic ungulate embryo is cultured, and wherein said culturing step comprises selection for one or more markers of said heterologous DNA molecule. 3. A method according to claim 1, wherein the transgenic ungulate is selected from the group consisting of a bovine, an ovine, a caprine, and a porcine. 4. A method according to claim 1, wherein the heterologous DNA molecule comprises one or more telomeres, one or more centromeres, and one or more origins of replication. 5. A method according to claim 1, wherein the heterologous DNA molecule is contained within the cells of the transgenic ungulate embryo on a replication unit that comprises essentially no homologous DNA. 6. A method according to claim 1, wherein said nuclear transfer embryo is cultured to at least the two cell stage, wherein at least 50% of the cells of the transgenic ungulate embryo comprise the heterologous DNA molecule. 7. A method according to claim 1, wherein the nuclear donor cell is selected from the group consisting of a somatic cell, a primordial germ cell, an embryonic germ cell, and an embryonic stem cell. 8. A method according to claim 1, wherein the heterologous DNA comprises a plurality of copies of at least one transgene. 9. A method according to claim 1, wherein said heterologous DNA molecule is between 100 kilobase pairs and 500 megabase pairs. 10. A method according to claim 1, wherein said heterologous DNA molecule is an artificial chromosome. 11. A method of producing a transgenic ungulate from a transgenic ungulate embryo of claim 1, the method comprising: transferring said transgenic ungulate embryo into a maternal host so as to produce a fetus of full fetal development and parturition to generate said transgenic ungulate. 12. A method according to claim 11, wherein prior to said transferring step, said transgenic ungulate embryo is cultured to at least the two cell stage. 13. A method according to claim 11, wherein at least 50% of the cells of the transgenic ungulate comprise the heterologous DNA molecule. 14. A method of producing a transgenic ungulate from a transgenic ungulate embryo of claim 1, the method comprising: (a) transferring said transgenic ungulate embryo into a maternal host so as to produce a fetus; (b) obtaining a nuclear donor cell from said fetus, wherein said cell comprises said heterologous DNA molecule; (c) fusing said nuclear donor cell, or a nucleus thereof, with an enucleated recipient cell of the same species as the said cell to form a second nuclear transfer embryo, wherein said second nuclear transfer embryo comprises said heterologous DNA molecule; (d) activating said second nuclear transfer embryo to provide a second transgenic ungulate embryo; and (e) transferring said second transgenic ungulate embryo into a maternal host so as to produce a fetus of full fetal development and parturition to generate said transgenic ungulate. 15. A method according to claim 14, wherein prior to said transferring step, said transgenic ungulate embryo and/or said second transgenic ungulate embryo is cultured to at least the two cell stage. 16. A method according to claim 14, wherein at least 50% of the cells of the transgenic ungulate comprise the heterologous DNA molecule. 17. A method of producing a transgenic ungulate from a transgenic ungulate embryo of claim 1, the method comprising: (a) transferring said transgenic ungulate embryo into a maternal host so as to produce a fetus; (b) obtaining one or more cells from said fetus, wherein one or more of said cells comprise said heterologous DNA molecule, and culturing said one or more cells to obtain a cell culture; (c) fusing a nuclear donor cell obtained from said cell culture, or a nucleus thereof, with an enucleated recipient cell of the same species as said nuclear donor cell to form a second nuclear transfer embryo, wherein said second nuclear transfer embryo comprises said heterologous DNA molecule; (d) activating said second nuclear transfer embryo to provide a second transgenic ungulate embryo; and (e) transferring said second transgenic ungulate embryo into a maternal host so as to produce a fetus of full fetal development and parturition to generate said transgenic ungulate. 18. A method according to claim 17, wherein prior to said transferring step, said transgenic ungulate embryo and/or said second transgenic ungulate embryo is cultured to at least the two cell stage. 19. A method according to claim 17, wherein at least 50% of the cells of the transgenic ungulate comprise the heterologous DNA molecule. 20. A method according to claim 17, wherein said culturing step comprises selection for one or more markers of said heterologous DNA molecule, whereby at least 90% of cells in said cell culture comprise said heterologous DNA molecule. 21. A transgenic ungulate embryo comprising one or more cells, wherein at least 50% of said one or more cells comprise an artificial chromosome. 22. A transgenic ungulate, wherein at least 50% of the cells making up said ungulate comprise an artificial chromosome. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention. Researchers have been developing methods for cloning mammalian animals over the past two decades. These reported methods typically include the steps of (1) isolating a pluripotent or totipotent cell; (2) inserting the cell or nucleus isolated from the cell into an enucleated oocyte (i.e., the oocyte's nucleus was previously extracted), and (3) allowing the embryo to mature in vivo. The first successful nuclear transfer experiment using mammalian cells was reported in 1983, when pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in like offspring(s). See, e.g., McGrath & Solter, 1983 , Science 220:1300-1302. Subsequently, other workers described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGCs). These cells are and can give rise to pluripotent cells (e.g., cells that can differentiate into other types of cells, and which may, but are not required to, differentiate into a grown animal). See, e.g., Matsui et al., 1992 , Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13:38. Progress has also been reported in the field of cloning ovine (sheep) animals (see, e.g., Willadsen, 1986, Nature 320:63-65; Campbell et al., 1996, Nature 380:64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature 385:810-813; PCT Publication WO 96/07732; PCT Publication WO 97/07668; and PCT publication WO 97/07669; and McCreath et al., 2000, Nature, 405:1066-1069), and bovine animals, (see, e.g., U.S. Pat. Nos. 4,994,384 and 5,057,420; Sims & First, 1993, Theriogenology 39:313; Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268; Delhaise et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994, J. Reprod. Dev. 37:413-424; Stice et al., 1996, Biol. Reprod. 54: 100-110; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells”). Researchers have also disclosed methods that resulted in cloned bovine animals (cattle). Bovines have been cloned using an embryonic cell derived from a 2-64 cell embryo as a nuclear donor. This bovine animal was reportedly cloned by utilizing nuclear transfer techniques set forth in U.S. Pat. Nos. 4,994,384 and 5,057,420. Others reported that cloned bovine embryos were formed where an inner cell mass cell of a blastocyst stage embryo was utilized as a nuclear donor in a nuclear transfer procedure (Sims & First, 1993, Theriogenology 39:313; Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268; and U.S. Pat. No. 6,107,543); a PGC isolated from fetal tissue as a nuclear donor (Delhaise et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994, J. Reprod. Dev. 37:413-424; and PCT application WO 95/10599 entitled “Embryonic Stem Cell-Like Cells”); a proliferating somatic cell (U.S. Pat. No. 5,945,577); and a reprogrammed nonembryonic cell (U.S. Pat. No. 6,011,197) Additionally, researchers have reported methods for obtaining cloned porcine animals and porcine chimeric animals, specifically, where a nuclear donor obtained from a 4-cell embryo is placed inside an enucleated zygote. See, e.g., Prather et al., 1989, Biology of Reproduction 41: 414-418; Piedrahita et al., 1998, Biology of Reproduction 58: 1321-1329; and WO 94/26884, “Embryonic Stem Cells for Making Chimeric and Transgenic Ungulates,” Wheeler, published Nov. 24, 1994. Also, researchers have reported nuclear transfer experiments using porcine nuclear donors and porcine oocytes. See., e.g., Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41: 414-419; Prather et al., 1990, Exp. Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259: 257-266; Terlouw et al., 1992, Theriogenology 37: 309, Pokajaeva et al., Nature 407, 86-90 (2000); Onishi et al., Science 289 1188-1190 (2000); and Betthauser et al., Nature Biotechnology 18: 1055-1059 (2000). Researchers have also developed methods for generating transgenic cells, which may be applicable to the production of transgenic animals. Although several viral vectors, non-viral vectors, and other delivery systems have been developed for establishing transgenic cells, many of these technologies are constrained by multiple limitations. Specifically, these limitations include (1) the size of inserted DNA is limited to approximately 10 kilobases (kb); (2) integration of the DNA of interest cannot be specifically targeted into the cell's nuclear DNA; and (3) expression of a recombinant product from the DNA of interest cannot be well controlled. See, e.g., Mitani et al., 1993, Trends Biotech, 11: 162-166; U.S. Pat. No. 5,633,067, “Method of Producing a Transgenic Bovine or Transgenic Bovine Embryo,” DeBoer et al, issued May 27, 1997; U.S. Pat. No. 5,612,205, “Homologous Recombination in Mammalian Cells,” Kay et al., issued Mar. 18, 1997; and PCT publication WO 93/22432, “Method for Identifying Transgenic Pre-Implantation Embryos,” all of which are incorporated by reference herein in their entirety, including all figures, drawings, and tables. Artificial chromosome technology is not constrained by the above-defined limitations. Moreover, researchers have discovered that artificial chromosomes can be replicated de novo. See, e.g., Kereso et al., 1996, Chromosome Research 4: 226-239, Holló et al., 1996, Chromosome Research 4: 240-247, U.S. Pat. No. 6,025,155, and U.S. Pat. No. 6,077,697. Each reference used to provide background information in this section is hereby incorporated by reference in its entirety, including ant tables, figures, and claims. Despite progress towards cloning mammals and establishing transgenic cells, there remains a great need in the art for materials and methods that enhance the efficiency for cloning transgenic animals. In particular, there remains a great need in the art to provide pluripotent and totipotent transgenic cells that can be utilized as nuclear donors. Furthermore, there remains a long felt need in the art for providing cell lines that are karyotypically stable and transgenic, which can be utilized in processes for cloning transgenic animals. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention relates in part to transgenic, totipotent, mammalian cells comprising one or more large, heterologous DNA constructs of 100 kbp or more. Preferably, the large DNA construct(s) are artificial chromosomes. Mammalian cells containing the large heterologous DNA construct(s) may be used for producing transgenic embryos and transgenic animals cloned from such cells. The invention is also directed in part to processes for producing totipotent cells that comprise one or more large, heterologous DNA constructs; processes for utilizing such cells; and processes for producing transgenic embryos and transgenic animals cloned from such cells. Thus, in a first aspect, the invention features a method for producing transgenic cells by inserting a large, heterologous DNA construct of 100 kbp or more into cells. Such cells may preferably be used as nuclear donor cells in methods to produce transgenic animals, most preferably ungulates. Preferably, a large, heterologous DNA construct is at least 200 Kbp, at least 300 Kbp, at least 400 Kbp, at least 500 Kbp, at least 750 Kbp, at least 1 Mbp, at least 5 Mbp, at least 10 Mbp, at least 20 Mbp, at least 50 Mbp, at least 100 Mbp, at least 500 Mbp, or at least 1000 Mbp. Particularly useful are artificial chromosomes of between 100 Kbp and 500 Mbp; between 500 Kbp and 500 Mbp; and between 1 Mbp and 500 Mbp. In certain embodiments, the large, heterologous DNA construct(s) of this aspect are artificial chromosomes. Advantages of using artificial chromosomes include: (1) target DNA greater than 10 kb can be inserted into cells; (2) the location of target DNA of interest can be controlled; (3) transgenic animals and embryos containing large foreign genes, or a large copy number of one or more foreign genes, in a majority of cells can be obtained; and (4) the expression levels of a recombinant product from the DNA of interest can be manipulated in vitro. Specifically, expression levels can be manipulated by controlling the copy number of target DNA and/or its regulation by promoters, enhancers, etc., in an artificial chromosome, as defined in greater detail hereafter. The term “artificial chromosome” as used herein refers to nucleic acid molecules that are generated by the manipulation of DNA, contain a centromere, and are capable of stable, autonomous replication in cells. An artificial chromosome (1) can replicate with naturally occurring chromosomes in the nucleus of target cell; (2) can be large in size (ranging in size from 100 kilobase pairs (Kbp) to 1000 megabase pairs (Mbp) in length, or more); (3) typically comprises a centromere, origins of replication, and telomeres; and (4) can comprise neutral DNA. Neutral DNA does not encode products that significantly alter the functions of a cell in which the artificial chromosome is located. For example, neutral DNA may encode ribosomal RNA. It is not typical that increasing levels of ribosomal RNA significantly alters cell functions. Neutral DNA can also be referred to as “satellite DNA.” Preferably, an artificial chromosome is at least 200 Kbp, at least 300 Kbp, at least 400 Kbp, at least 500 Kbp, at least 750 Kbp, at least 1 Mbp, at least 5 Mbp, at least 10 Mbp, at least 20 Mbp, at least 50 Mbp, at least 100 Mbp, at least 500 Mbp, or at least 1000 Mbp. Particularly useful are artificial chromosomes of between 100 Kbp and 500 Mbp; between 500 Kbp and 500 Mbp; and between 1 Mbp and 500 Mbp. Materials and methods for producing, identifying, and characterizing artificial chromosomes are well known in the art. See, e.g., Kereso et al., 1996, Chromosome Research 4: 226-239, Holló et al., 1996, Chromosome Research 4: 240-247, International publication nos. WO00/18941, WO98/08964, WO97/16533 and WO97/40183, and U.S. Pat. Nos. 5,721,118, 6,025,155, 6,077,697, and 6,133,503, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings. These publications also describe shuttle vectors useful for incorporating target DNA into artificial chromosomes. Artificial chromosomes can arise from a portion of a natural chromosome by manipulation. Artificial chromosomes can be detected in cells by using chromosome identification techniques well known in the art. An example of such a technique is chromosome karyotype analysis. Mammalian artificial chromosomes (MACs) can be generated by cellular mediated chromosome assembly from transfected alphoid, telomeric and marker DNAs (Harrington J. J. et al. Nature Genetics, 15, 345-355, 1997; Ikeno, M. et al, Nature Biotechnology 16, 431-439, 1998; Henning, K. A. et al, PNAS USA 96, 592-597, 1999) and even from non-alphoid DNA (du Sart D, et al, Nature Genetics 16, 144-153, 1997). Minichromosomes may be generated by fragmenting natural human chromosomes using telomere-directed breakage (Shen M H, et al, Human Molecular Genetics 6, 1375-1382, 1997; Shen M H et al, Current Biology 10, 31-34, 1999). It is possible to transfer human-murine minichromosome chimeras (Shen M H et al, Current Biology 10, 31-34, 1999), fragmented human minichromosomes Tomizuka K et al, Nature Genetics 16, 133-143, 1997; Tomizuka K et al, PNAS USA 97, 722-797, 2000), and human small accessory chromosomes (SACs; Vermeesch J R et al, Human Genetics 105, 611-618, 1999) via microcell-mediated chromosome transfer (MMCT) to recipient cells. The term “target DNA” as used herein refers to DNA that is intended to be or has been incorporated into a large heterologous DNA construct, preferably an artificial chromosome. The term “heterologous” is defined below. Target DNA can encode multiple types of recombinant products, as defined hereafter, and may exist in multiple copies when introduced into an artificial chromosome. One advantage of artificial chromosome technology is that target DNA copy number can be controlled and monitored in an artificial chromosome in vitro before the artificial chromosome comprising the target DNA is introduced into a cell. In addition, depending on the promoter used, expression can also be monitored in vitro. This advantage is contrasted with many existing techniques for creating transgenic cells, which cause random insertion of target DNA into a cell nuclear DNA. Materials and methods for introducing target DNA into an artificial chromosome and materials and methods for introducing the resulting artificial chromosome into cells are defined hereafter. The term “heterologous nucleic acid” refers to nucleic acids having (1) a nucleic acid sequence that differs from the nucleic acid sequences present in cell's naturally occurring nuclear DNA; (2) a subset of nucleic acid having a nucleotide sequence that is present in cell nuclear DNA, but that exists in different proportions in the heterologous nucleic acid than in cell nuclear DNA; (3) a nucleic acid sequence originating from a species other than the species from which cell nuclear DNA originates; and (4) a nucleic acid sequence that differs from the DNA sequences present in cell's naturally occurring mitochondrial DNA. Artificial chromosomes, such as mammalian artificial chromosomes [MACs], can be generated and isolated by the methods described in the publications above. In particularly preferred embodiments, two types of artificial chromosomes are used, both of which function in cells as stable, functional chromsomes. One type, herein referred to as ACEs (“Artificial Chromosome Expression systems” based on satellite DNA) is a stable heterochromatic chromosome, and the other type is a de novo-formed minichromosomes based on amplification of euchromatin. Artificial chromosomes, and, in particular the two preferred types discussed above, provide an extra-genomic locus for targeted integration of up to multi-megabase pair size DNA fragments that contain single or multiple genes, including multiple copies of a single gene operatively linked to one promoter or each copy or several copies linked to separate promoters. Thus, methods provided can be used to introduce genes via MACs into cells and tissues of ungulate mammals. The artificial chromosomes with integrated heterologous DNA may be used in methods of production of gene products, particularly products that require expression of multigenic biosynthetic pathways, and also are intended for delivery into the nuclei of cells, such as nuclear donor cells used in nuclear transfer procedures, for production of transgenic ungulate mammals. Additionally, such artificial chromosomes provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit up to multi-megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway, a very large gene such as the cystic fibrosis transmembrane conductance regulator gene (approximately 250 kb genomic DNA gene), or several genes, such as multiple genes encoding a series of antigens for preparation of a multivalent vaccine, can be stably introduced into a cell. The artificial chromosomes described herein, including ACEs and euchromatin-based minichromosomes, can be generated by introducing heterologous DNA, preferably including DNA encoding one or multiple selectable marker(s), into cells, preferably a stable cell line, growing the cells under selective conditions, and identifying from among the resulting cell clones those that include chromosomes with more than one centromere, fragments thereof, and/or heterochromatic structures. Amplification that produces the additional centromere(s) occurs in cells that contain chromosomes in which heterologous DNA has integrated near the centromere in the pericentric region of the chromosome. Selected cells comprising intermediates in the formation of such artificial chromosomes can then be used to generate complete artificial chromosomes. For example, continued culture of cells containing a formerly dicentric chromosome under conditions that destabilize chromosomes (such as BrdU treatment) and/or under selective conditions can yield ACEs. Similarly, artificial chromosomes can be generated by culturing cells with multi-centric (typically dicentric) chromosomes under conditions whereby the chromosome breaks to form a minichromosome and a formerly dicentric chromosome. Among the MACs provided herein can be ACEs, which are predominantly heterochromatic (i.e., contain more heterochromatin than euchromatin, and preferably contain about 70% heterochromatin), and can comprise repeating units of short satellite DNA, so that without insertion of heterologous or foreign DNA, the chromosomes preferably contain no genetic information. They can thus be used as “safe” vectors for delivery of DNA to mammalian hosts because they do not contain any potentially harmful genes. ACEs are generated, not from the minichromosome fragment as, for example, in U.S. Pat. No. 5,288,625 (which is incorporated herein by reference in its entirety including all figures, tables, and drawings), but from the fragment of the formerly dicentric chromosome. In addition, euchromatic minichromosomes can be generated. Methods for generating one type of MAC, the minichromosome, is described in U.S. Pat. No. 5,288,625 (which is incorporated herein by reference in its entirety including all figures, tables, and drawings), along with its use for the expression of heterologous DNA are provided. In preferred embodiments, (1) the artificial chromosome is an ACEs comprising one or more markers; (2) a marker is an antibiotic resistance gene selected from the group consisting of neomycin resistance gene, hygromycin resistance gene, and puromycin resistance gene; (3) the artificial chromosome comprises a DNA sequence that encodes one or more recombinant products; (4) a recombinant product is a ribozyme; (5) a recombinant product is antisense RNA; (6) a recombinant product is a peptide; (7) a recombinant product is a polypeptide; (8) a recombinant product is a protein; (9) a recombinant product is an enzyme; (10) a recombinant product is expressed in a biological fluid; (11) a recombinant product is expressed in a tissue; (12) a recombinant product confers resistance to one or more parasites and/or diseases; (13) an artificial chromosome comprises one or more regulatory elements; (14) a regulatory element is a promoter element; (15) a promoter element is selected from the group consisting of milk protein promoter, urine protein promoter, blood protein promoter, tear duct protein promoter, synovial protein promoter, mandibular gland protein promoter, casein promoter, β-casein promoter, melanocortin promoter, milk serum protein promoter, α-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and α-actin promoter; (17) a regulatory element is a repressor element; (18) a regulatory element is an insulator element; and (19) a regulatory element is an enhancer element. The term “marker” as used herein refers to any DNA sequence that distinguishes a cell comprising an artificial chromosome, or a precursor thereof, from a cell that does not comprise the artificial chromosome or precursor. For example, a marker can be used in the initial steps of generating ACEs, whereby the marker distinguishes a cell containing a foreign nucleic acid from a cell that does not contain the foreign nucleic acid. Multiple types of markers, such as genes encoding green fluorescent protein, antibiotic resistance, β-galactosidase, glutamine synthetase, thymidine kinase, cytosine deaminase, and dihydrofolate reductase are well known in the art. Preferred as markers are DNA sequences that encode a molecule which directly or indirectly inactivates a drug that retards the growth of cells not expressing such a molecule. Examples of these latter described markers are blasticidin-S, neomycin, hygromycin, and puromycin resistance genes. These examples are not meant to be limiting and the invention relates in part to any marker known in the art. The term “ribozyme” as used herein refers to ribonucleic acid molecules that can cleave other RNA molecules in specific regions. Ribozymes can bind to discrete regions on a RNA molecule, and then specifically cleave a region within that binding region or adjacent to the binding region. Ribozyme techniques can thereby decrease the amount of polypeptide translated from formerly intact message RNA molecules. For specific descriptions of ribozymes, see U.S. Pat. No. 5,354,855, entitled “RNA Ribozyme which Cleaves Substrate RNA without Formation of a Covalent Bond,” Cech et al., issued on Oct. 11, 1994, and U.S. Pat. No. 5,591,610, entitled “RNA Ribozyme Polymerases, Dephosphorylases, Restriction Endoribonucleases and Methods,” Cech et al., issued on Jan. 7, 1997, both of which are incorporated by reference in their entireties including all figures, tables, and drawings. The term “antisense RNA” as used herein refers to any RNA that binds to mRNA with enough affinity to decrease the amount of protein translated from the mRNA. The amount of protein translated from the mRNA is preferably decreased by more than 20%; more preferably decreased by more than 50%, 70%, and 80%; and most preferably decreased by more than 90%. Antisense RNA materials and methods are well known in the art. The terms “biological fluid” and the term “tissue” as used herein refer to any fluid or tissue in or from a biological organism. The fluids may include, but are not limited to, tears, saliva, milk, urine, amniotic fluid, semen, plasma, oviductal fluid, and synovial fluid. The tissues may include, but are not limited to, lung, heart, blood, liver, muscle, brain, pancreas, skin, and others. The term “confers resistance” as used herein refers to the ability of a recombinant product to completely abrogate or partially alleviate the symptoms of a disease or parasitic condition. Hence, if a disease is related to inflammation, for example, a recombinant product can confer resistance to that inflammation if the inflammation decreases upon expression of the recombinant product. A recombinant product may confer resistance or partially confer resistance to a disease or parasitic condition, for example, if the recombinant product is an antisense RNA molecule that specifically binds to an mRNA molecule encoding a polypeptide responsible for the inflammation. In preferred embodiments, the DNA with the selectable marker that is introduced into cells to generate artificial chromosomes includes sequences that target it to the pericentric region of the chromosome. Integration of the DNA into existing chromosomes in the cells can induce amplification that results in generation of additional centromeres. Transgenic Cells Large heterologous nucleic acid constructs, such as artificial chromosomes, can then be introduced into cells to produce stable transformed cell lines and cells. Introduction is effected by any suitable method or combination of methods including, but not limited to microinjection, cell fusion, microcell fusion, electroporation, sonoporation, electrofusion, projectile bombardment, calcium phosphate precipitation, lipid-mediated transfer systems, ligand/receptor systems and other such methods well known to the skilled artisan. ACEs in particular can be readily isolated and used for gene delivery. These artificial chromosomes can also be used in gene product production systems, production of humanized genetically transformed animal organs, and, most preferably, the generation of transgenic ungulates. In certain embodiments, the invention relates to transgenic, totipotent, mammalian cells comprising at least one artificial chromosome, but the invention relates in part to any number of artificial chromosomes in a totipotent mammalian cell. A totipotent mammalian cell preferably comprises ten or fewer artificial chromosomes; more preferably comprises six or fewer artificial chromosomes, four or fewer artificial chromosomes, or two or fewer artificial chromosomes; and most preferably comprises one artificial chromosome. If a totipotent mammalian cell of the invention comprises more than one artificial chromosome, the artificial chromosomes may be identical or may differ from one another. The term “transgenic” as used herein in reference to cells refers to a cell that comprises heterologous nucleic acid, preferably deoxyribonucleic acid (DNA). In preferred embodiments, a transgenic cell comprises one or more heterologous DNA sequences. In other preferred embodiments, a transgenic cell is a cell in which one or more endogenous genes have been deleted, duplicated, activated, or modified. In particularly preferred embodiments, a transgenic cell comprises both one or more heterologous DNA sequences, and one or more endogenous genes that have been deleted, duplicated, activated, or modified. An artificial chromosome present in a transgenic cell can comprise heterologous DNA. Heterologous DNA can encode multiple types of recombinant products, as defined hereafter. The term “transgene” as used herein refers to a single gene that is partially or entirely transgenic in origin. In certain embodiments, greater than 50% of the transgene consists of heterologous DNA. In preferred embodiments, greater than 75% of the transgene consists of heterologous DNA, greater than 80% of the transgene consists of heterologous DNA, greater than 90% of the transgene consists of heterologous DNA, greater than 95% of the transgene consists of heterologous DNA, greater than 98% of the transgene consists of heterologous DNA, and 100% of the transgene consists of heterologous DNA. The term “different nucleic acid sequence” as used herein refers to nucleic acid sequences that are not substantially similar. The term “substantially similar” as used herein in reference to nucleic acid sequences refers to two nucleic acid sequences having preferably 80% or more nucleic acid identity, more preferably 90% or more nucleic acid identity or most preferably 95% or more nucleic acid identity. Nucleic acid identity is a property of nucleic acid sequences that measures their similarity or relationship when aligned by means known to one skilled in the art. Identity is measured by dividing the number of identical bases in the two sequences by the total number of bases and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity and similarity using standard parameters, for example Gapped BLAST or PSI-BLAST (Altschul, et al. (1997) Nucleic Acids Res. 25:3389-3402), BLAST (Altschul, et al. (1990) J. Mol. Biol. 215:403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol. 147:195-197). Preferably, the default settings of these programs will be employed, but those skilled in the art recognize whether these settings need to be changed and know how to make the changes. The term “substantially similar” as used herein in reference to amino acid sequences refers to two amino acid sequences having preferably 50% or more amino acid identity, more preferably 70% or more amino acid identity or most preferably 90% or more amino acid identity. Amino acid identity is a property of amino acid sequence that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. “Similarity” in protein sequences is measured by dividing the number of identical residues plus the number of conservatively substituted residues (see Bowie, et al. Science, (1999) 247, 1306-1310, which is incorporated herein by reference in its entirety, including any drawings, figures, or tables) by the total number of residues and gaps and multiplying the product by 100. “Similarity” in nucleic acid sequences is measured by dividing the number of identical bases by the total number of residues and gaps and multiplying the product by 100. The term “recombinant product” as used herein refers to the product produced from a target DNA sequence. A recombinant product can be a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a polypeptide that binds to a regulatory element (a term described hereafter), a structural protein, an RNA molecule, and/or a ribozyme, for example. These products are well defined in the art. This list of products is for illustrative purposes only and the invention relates to other types of recombinant products. In preferred embodiments, (1) the mammalian cell is an ungulate cell; (2) the ungulate is selected from the group consisting of bovids, ovids, cervids, suids, equids and camelids; (3) the ungulate is bovine; (4) the mammalian cell is a nonembryonic cell; (5) the mammalian cell is a fetal cell; and (6) the mammalian cell is an adult cell. The term “mammalian” as used herein refers to any animal of the class Mammalia. Preferably, a mammalian cell or cell line is a placental, a monotreme and a marsupial. Most preferably, a mammalian cell or cell line is a canid, felid, murid, leporid, ursid, mustelid, ungulate, ovid, suid, equid, bovid, caprid, cervid, and a human or non-human primate. A mammalian cell or cell line can be isolated from any source of mammalian cells including, but not limited to, a mammalian embryo, a mammalian fetus, and a mammalian animal. The term “canid” as used herein refers to any animal of the family Canidae. Preferably, a canid cell or cell line is isolated from a wolf, a jackal, a fox, and a domestic dog. The term “felid” as used herein refers to any animal of the family Felidae. Preferably, a felid cell or cell line is isolated from a lion, a tiger, a leopard, a cheetah, a cougar, and a domestic cat. The term “murid” as used herein refers to any animal of the family Muridae. Preferably, a murid cell or cell line is isolated from a mouse and a rat. The term “leporid” as used herein refers to any animal of the family Leporidae. Preferably, a leporid cell or cell line is isolated from a rabbit. The term “ursid” as used herein refers to any animal of the family Ursidae. Preferably, a ursid cell or cell line is isolated from a bear. The term “mustelid” as used herein refers to any animal of the family Mustelidae. Preferably, a mustelid cell or cell line is isolated from a weasel, a ferret, an otter, a mink, and a skunk. The term “primate” as used herein refers to any animal of the Primate order. Preferably, a primate cell or cell line is isolated from an ape, a monkey, a chimpanzee, and a lemur. The term “ungulate” as used herein refers to any animal of the polyphyletic group formerly known as the taxon Ungulata. Preferably, an ungulate cell or cell line is isolated from a camel, a hippopotamus, a horse, a tapir, and an elephant. Most preferably, an ungulate cell or cell line is isolated from a sheep, a cow, a goat, and a pig. Especially preferred in the bovine species are Bos taurus, Bos indicus , and Bos buffaloes cows or bulls. The term “ovid” as used herein refers to any animal of the family Ovidae. Preferably, an ovid cell or cell line is isolated from a sheep. The term “suid” as used herein refers to any animal of the family Suidae. Preferably, a suid cell or cell line is isolated from a pig or a boar. The term “equid” as used herein refers to any animal of the family Equidae. Preferably, an equid cell or cell line is isolated from a zebra or an ass. Most preferably, an equid cell or cell line is isolated from a horse. The term “bovid” as used herein refers to any animal of the family Bovidae. Preferably, an bovid cell or cell line is isolated from an antelope, an oxen, a cow, and a bison. The term “caprid” as used herein refers to any animal of the family Caprinae. Preferably, a caprid cell or cell line is isolated from a goat. The term “cervid” as used herein refers to any animal of the family Cervidae. Preferably, a cervid cell or cell line is isolated from a deer. The term “immortalized” or “permanent” as used herein in reference to cells refers to cells that have exceeded the Hayflick limit. The Hayflick limit can be defined as the number of cell divisions that occur before a cell line becomes senescent. Hayflick set this limit to approximately 60 divisions for most non-immortalized cells. See, e.g., Hayflick and Moorhead, 1961, Exp. Cell. Res. 25: 585-621; and Hayflick, 1965, Exp. Cell Research 37: 614-636, incorporated herein by reference in their entireties including all figures, tables, and drawings. Therefore, an immortalized cell line can be distinguished from non-immortalized cell lines if the cells in the cell line are able to undergo more than 60 divisions. If the cells of a cell line are able to undergo more than 60 cell divisions, the cell line is an immortalized or permanent cell line. The immortalized cells of the invention are preferably able to undergo more than 70 divisions, are more preferably able to undergo more than 80 divisions, and are most preferably able to undergo more than 90 cell divisions. The terms “primary culture” and “primary cell” refer to cells taken from a tissue source, and their progeny, grown in culture before subdivision and transfer to a subculture. The terms “plated” or “plating” as used herein in reference to cells refer to establishing cell cultures in vitro. For example, cells can be diluted in cell culture media and then added to a cell culture plate or cell culture dish. Cell culture plates are commonly known to a person of ordinary skill in the art. Cells may be plated at a variety of concentrations and/or cell densities. In preferred embodiments, plated cells may grow to confluence. The meaning of the term “cell plating” can also extend to the term “cell passaging.” Cells of the invention can be passaged using cell culture techniques well known to those skilled in the art. The term “cell passaging” refers to such techniques which typically involve the steps of (1) releasing cells from a solid support and disassociation of these cells, and (2) diluting the cells in fresh media suitable for cell proliferation. Immortalized cells can be successfully grown by plating the cells in conditions where they lack cell to cell contact. Cell passaging may also refer to removing a portion of liquid medium bathing cultured cells and adding liquid medium from another source to the cell culture to dilute the cell concentration. The term “proliferation” as used herein in reference to cells refers to a group of cells that can increase in size and/or can increase in numbers over a period of time. The term “confluence” as used herein refers to a group of cells where a large percentage of the cells are physically contacted with at least one other cell in that group. Confluence may also be defined as a group of cells that grow to a maximum cell density in the conditions provided. For example, if a group of cells can proliferate in a monolayer and they are placed in a culture vessel in a suitable growth medium, they are confluent when the monolayer has spread across a significant surface area of the culture vessel. The surface area covered by the cells preferably represents about 50% of the total surface area, more preferably represents about 70% of the total surface area, and most preferably represents about 90% of the total surface area. The cells and cell lines of the instant invention may be cultured. The term “cultured” as used herein in reference to cells refers to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment. An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example. Specific examples of suitable in vitro environments for cell cultures are described in Culture of Animal Cells: a manual of basic techniques (3 rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells : a laboratory manual (vol. 1), 1998; D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan John Wiley and Sons, Ltd., each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings. Cells may be cultured in suspension and/or in monolayers with one or more substantially similar cells. Cells may be cultured in suspension and/or in monolayers with a heterogeneous population of cells. The term “heterogeneous” as utilized in the previous sentence can relate to any cell characteristics, such as cell type and cell cycle stage, for example. Cells may be cultured in suspension, cultured as monolayers attached to a solid support, and/or cultured on a layer of feeder cells, for example. The term “feeder cells” is defined hereafter. Furthermore, cells may be successfully cultured by plating the cells in conditions where they lack cell to cell contact. Preferably, cultured cells undergo cell division and are cultured for at least 5 days, more preferably for at least 10 days or 20 days, and most preferably for at least 30 days. Preferably, a significant number of cultured cells do not terminate while in culture. The terms “terminate” and “significant number” are defined hereafter. Nearly any type of cell can be placed in cell culture conditions. Cultured cells can be utilized to establish a cell line. In particularly preferred embodiments, a cell may be “clonally propagated.” In these embodiments, cells are diluted to an extent such that, statistically, some or all of the culture vessels into which the diluted cells are placed will contain only a single cell. Thus, the culture that grows within these culture vessels will be derived from a single cell. Materials and methods for clonally propagating cells are described in U.S. patent application Ser. No. 09/753,323 (attorney docket number 030653.0026.CIP1, filed Dec. 28, 2000), which is hereby incorporated in its entirety. The term “terminating” and “terminate” as used herein with regard to cultured cells may refer to cells that undergo cell death, which can be measured using multiple techniques known to those skilled in the art (e.g., CytoTox96® Cytotoxicity Assay, Promega, Inc. catalog no. G1780; Celltiter96® Aqueous Cell Proliferation Assay Kit, Promega, Inc. catalog no. G3580; and Trypan Blue solution for cytotoxicity assays, Sigma catalog no. T6146). Termination may also be a result of apoptosis, which can be measured using multiple techniques known to persons skilled in the art (e.g., Dead End™ Apoptosis Detection Kit, Promega, Inc. catalog no. G7130). Terminated cells may be identified as those that have undergone cell death and/or apoptosis and have released from a solid surface in culture. In addition, terminated cells may lack intact membranes which can be identified by procedures described above. Also, terminated cells may exhibit decreased metabolic activity, which may be caused in part by decreased mitochondrial activity that can be identified by rhodamine 1,2,3, for example. Furthermore, termination can be refer to cell cultures where a significant number of cultured cells terminate. The term “significant number” in the preceding sentence refers to about 80% of the cells in culture, preferably about 90% of the cells in culture, more preferably about 100% of the cells in culture, and most preferably 100% of the cells in culture. The term “suspension” as used herein refers to cell culture conditions in which the cells are not attached to a solid support. Cells proliferating in suspension can be stirred while proliferating using apparatus well known to those skilled in the art. The term “monolayer” as used herein refers to cells that are attached to a solid support while proliferating in suitable culture conditions. A small portion of the cells proliferating in the monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support. Preferably less than 15% of these cells are not attached to the solid support, more preferably less than 10% of these cells are not attached to the solid support, and most preferably less than 5% of these cells are not attached to the solid support. The term “substantially similar” as used herein in reference to mammalian cells refers to cells from the same organism and the same tissue. Substantially similar can also refer to cell populations that have not significantly differentiated. For example, preferably less than 15% of the cells in a population of cells have differentiated, more preferably less than 10% of the cell population have differentiated, and most preferably less than 5% of the cell population have differentiated. The term “cell line” as used herein refers to cultured cells that can be passaged one or more times. The invention preferably relates to cell lines that can be passaged more than 2, 5, 10, 15, 20, 30, 50, 80, 100, and 200 times. The concept of cell passaging is defined previously. In preferred embodiments, (1) the mammalian cell is subject to manipulation; (2) the manipulation comprises the step of nuclear transfer; (3) the nuclear transfer comprises the step of inserting the totipotent mammalian cell into a recipient oocyte; (4) the manipulation comprises a step of cryopreservation of the mammalian cell; (5) the manipulation comprises a step of thawing of the mammalian cell; (6) the manipulation comprises a step of culturing the mammalian cell; (7) the manipulation comprises a step of passaging the mammalian cell; (8) the manipulation comprises a step of synchronizing the mammalian cell; (9) the manipulation comprises a step of introducing the mammalian cell to feeder cells; and (10) the manipulation comprises a step of dissociating the mammalian cell from other cells. The term “manipulation” as used herein refers to the common usage of the term, which is the management or handling directed towards some object. Examples of manipulations are described herein. The term “thawing” as used herein refers to the process of increasing the temperature of a cryopreserved cell, embryo, or portions of animals. Methods of thawing cryopreserved materials such that they are active after the thawing process are well-known to those of ordinary skill in the art. The term “dissociating” as used herein refers to the materials and methods useful for pulling a cell away from another cell. For example, a blastomere (i.e., a cellular member of a morula or blastocyst stage embryo) can be pulled away from the rest of the developing cell mass by techniques and apparatus well known to a person of ordinary skill in the art. See, e.g., U.S. Pat. No. 4,994,384, entitled “Multiplying Bovine Embryos,” issued on Feb. 19, 1991, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. Alternatively, cells proliferating in culture can be separated from one another to facilitate such processes as cell passaging, which is described previously. In addition, dissociation of a cultured cell from a group of cultured cells can be useful as a first step in the process of nuclear transfer, as described hereafter. When a cell is dissociated from an embryo, the dissociation manipulation can be useful for such processes as re-cloning, a process described herein, as well as a step for multiplying the number of embryos. The term “non-embryonic cell” as used herein refers to a cell that is not isolated from an embryo. Non-embryonic cells can be differentiated or non-differentiated. Non-embryonic cells refers to nearly any somatic cell, such as cells isolated from an ex utero animal. These examples are not meant to be limiting. The term “fetus” as used herein refers to a developing cell mass that has implanted into the uterine membrane of a maternal host. A fetus can include such defining features as a genital ridge, for example. A genital ridge is a feature easily identified by a person of ordinary skill in the art, and is a recognizable feature in fetuses of most animal species. The term “fetal cell” as used herein refers to any cell isolated from and/or has arisen from a fetus or derived from a fetus. The term “non-fetal cell” is a cell that is not derived or isolated from a fetus. When cells are isolated from a fetus, such cells are preferably isolated from fetuses where the fetus is between 20 days and parturition, between 30 days and 100 days, more preferably between 35 days and 70 days and between 40 days and 60 days, and most preferably about a 55 day fetus. An age of a fetus can be determined from the time that an embryo, which develops into the fetus, is established. The term “about” with respect to fetuses refers to plus or minus five days. The term “parturition” as used herein refers to a time that a fetus is delivered from female recipient. A fetus can be delivered from a female recipient by abortion, c-section, or birth. In preferred embodiments, the cells and cell lines of the instant invention are primary cells, embryonic cells, non-embryonic cells, fetal cells, genital ridge cells, primordial germ cells, embryonic germ cells, embryonic stem cells, somatic cells, adult cells, fibroblasts, differentiated cells, undifferentiated cells, amniotic cells, ovarian follicular cells, and cumulus cells. Preferably, such cells grow to confluent monolayers in culture. The term “primordial germ cell” as used herein refers to a diploid somatic cell capable of becoming a germ cell. Primordial germ cells can be isolated from the genital ridge of a developing cell mass. The genital ridge is a section of a developing cell mass that is well-known to a person of ordinary skill in the art. See, e.g., Strelchenko, 1996, Theriogenology 45: 130-141 and Lavoir 1994, J. Reprod. Dev. 37: 413-424. Such cells, when cultured, are referred to by the skilled artisan as “embryonic germ cells.” The term “embryonic stem cell” as used herein refers to pluripotent cells isolated from an embryo that are maintained in in vitro cell culture. Embryonic stem cells may be cultured with or without feeder cells. Embryonic stem cells can be established from embryonic cells isolated from embryos at any stage of development, including blastocyst stage embryos and pre-blastocyst stage embryos. Embryonic stem cells are well known to a person of ordinary skill in the art. See, e.g., WO 97/37009, entitled “Cultured Inner Cell Mass Cell-Lines Derived from Ungulate Embryos,” Stice & Golueke, published Oct. 9, 1997, and Yang & Anderson, 1992, Theriogenology 38: 315-335, both of which are incorporated herein by reference in their entireties, including all figures, tables, and drawings. The term “differentiated cell” as used herein refers to a cell that has developed from an unspecialized phenotype to that of a specialized phenotype. For example, embryonic cells can differentiate into an epithelial cell lining the intestine. It is highly unlikely that differentiated cells revert into their precursor cells in vivo or in vitro. However, materials and methods of the invention can reprogram differentiated cells into immortalized, totipotent cells. Differentiated cells can be isolated from a fetus or a live born animal, for example. The term “undifferentiated cell” as used herein refers to a cell that has an unspecialized phenotype and is capable of differentiating. An example of an undifferentiated cell is a stem cell. The term “asynchronous population” as used herein refers to cells that are not arrested at any one stage of the cell cycle. Many cells can progress through the cell cycle and do not arrest at any one stage, while some cells can become arrested at one stage of the cell cycle for a period of time. Some known stages of the cell cycle are G 0 , G 1 , S, G 2 , and M. An asynchronous population of cells is not manipulated to synchronize into any one or predominantly into any one of these phases. Cells can be arrested in the G 0 stage of the cell cycle, for example, by utilizing multiple techniques known in the art, such as by serum deprivation. Examples of methods for arresting non-immortalized cells in one part of the cell cycle are discussed in WO 97/07669, entitled “Quiescent Cell Populations for Nuclear Transfer,” hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. The terms “synchronous population” and “synchronizing” as used herein refer to a fraction of cells in a population that are arrested (i.e., the cells are not dividing) in a discrete stage of the cell cycle. Preferably, about 50% of the cells in a population of cells are arrested in one stage of the cell cycle, more preferably about 70% of the cells in a population of cells are arrested in one stage of the cell cycle, and most preferably about 90% of the cells in a population of cells are arrested in one stage of the cell cycle. Cell cycle stage can be distinguished by relative cell size as well as by a variety of cell markers well known to a person of ordinary skill in the art. For example, cells can be distinguished by such markers by using flow cytometry techniques well known to a person of ordinary skill in the art. Alternatively, cells can be distinguished by size utilizing techniques well known to a person of ordinary skill in the art, such as by the utilization of a light microscope and a micrometer, for example. The term “adult cell” as used herein refers to a cell from a live-born animal. The term “amniotic cell” as used herein refers to any cultured or non-cultured cell isolated from amniotic fluid. Examples of methods for isolating and culturing amniotic cells are discussed in Bellow et al., 1996, Theriogenology 45: 225; Garcia & Salaheddine, 1997, Theriogenology 47: 1003-1008; Leibo & Rail, 1990, Theriogenology 33: 531-552; and Vos et al., 1990, Vet. Rec. 127: 502-504, each of which is incorporated herein by reference in its entirety, including all figures tables and drawings. Particularly preferred are cultured amniotic cells that are rounded (e.g., cultured amniotic cells that do not display a fibroblast-like morphology). Also preferred amniotic cells are fetal fibroblast cells. The terms “fibroblast,” fibroblast-like,” “fetal,” and “fetal fibroblast” are defined hereafter. The term “fibroblast” as used herein refers to cultured cells having a flattened and elongated morphology that are able to grow in monolayers. Preferably, fibroblasts grow to confluent monolayers in culture. While fibroblasts characteristically have a flattened appearance when cultured on culture media plates, fetal fibroblast cells can also have a spindle-like morphology. Fetal fibroblasts may require density limitation for growth, may generate type I collagen, and may have a finite life span in culture of approximately fifty generations. Preferably, fetal fibroblast cells rigidly maintain a diploid chromosomal content. For a description of fibroblast cells, see, e.g., Culture of Animal Cells: a manual of basic techniques (3 rd edition), 1994, R. I. Freshney (ed), Wiley-Liss, Inc., incorporated herein by reference in its entirety, including all figures, tables, and drawings. The term “uterine cell” as used herein refers to any cell isolated from a uterus. Preferably, a uterine cell is a cell deriving from a pregnant adult animal. In preferred embodiments, uterine cells are cells obtained from fluid that fills the uterine cavity. Such cells can be obtained by numerous methods well known in the art such as amniocentesis. The term “ovarian follicular cell” as used herein refers to a cultured or non-cultured cell obtained from an ovarian follicle, other than an oocyte. Follicular cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles. Furthermore, follicular cells may be isolated when an oocyte in an ovarian follicle is immature (i.e., an oocyte that has not progressed to metaphase II) or when an oocyte in an ovarian follicle is mature (i.e., an oocyte that has progressed to metaphase II or a later stage of development). Preferred follicular cells include, but are not limited to, pregranulosa cells, granulosa cells, theca cells, columnar cells, stroma cells, theca interna cells, theca externa cells, mural granulosa cells, luteal cells, and corona radiata cells. Particularly preferred follicular cells are cumulus cells. Various types of follicular cells are known and can be readily distinguished by those skilled in the art. See, e.g., Laboratory Production of Cattle Embryos, 1994, Ian Gordon, CAB International; Anatomy and Physiology of Farm Animals (5th ed.), 1992, R. D. Frandson and T. L. Spurgeon, Lea & Febiger, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables. Individual types of follicular cells may be cultured separately, or a mixture of types may be cultured together. The term “cumulus cell” as used herein refers to any cultured or non-cultured cell isolated from cells and/or tissue surrounding an oocyte. Persons skilled in the art can readily identify cumulus cells. Examples of methods for isolating and/or culturing cumulus cells are discussed in Damiani et al., 1996, Mol. Reprod. Dev. 45: 521-534; Long et al., 1994, J: Reprod. Fert. 102: 361-369; and Wakayama et al., 1998, Nature 394: 369-373, each of which is incorporated herein by reference in its entireties, including all figures, tables, and drawings. Cumulus cells may be isolated from ovarian follicles at any stage of development, including primordial follicles, primary follicles, secondary follicles, growing follicles, vesicular follicles, maturing follicles, mature follicles, and graafian follicles. Cumulus cells may be isolated from oocytes in a number of manners well known to a person of ordinary skill in the art. For example, cumulus cells can be separated from oocytes by pipeting the cumulus cell/oocyte complex through a small bore pipette, by exposure to hyaluronidase, or by mechanically disrupting (e.g. vortexing) the cumulus cell/oocyte complex. Additionally, exposure to Ca ++ /Mg ++ free media can remove cumulus from immature oocytes. Also, cumulus cell cultures can be established by placing matured oocytes in cell culture media. Once cumulus cells are removed from media containing increased LH/FSH concentrations, they can to attach to the culture plate. In a preferred embodiment, the culturing process can comprise the step of selecting totipotent mammalian cells comprising at least one artificial chromosome. The term “selection” as used herein refers to a process for identifying cells that comprise a large heterologous nucleic acid construct, such as an artificial chromosome. Selection can be effected by identifying a marker region incorporated in an artificial chromosome. The term “marker” is defined previously. Preferably, from 50% to 100% of cells in cell cultures that have undergone selection comprise an artificial chromosome. In particularly preferred embodiments, greater than or equal to 50% of cells in cell cultures that have undergone selection comprise an artificial chromosome. More preferably, greater than or equal to 75% of cells in cell cultures that have undergone selection comprise an artificial chromosome. Most preferably, greater than or equal to 90% of cells in cell cultures that have undergone selection comprise an artificial chromosome. The term “feeder cells” as used herein refers to cells grown in co-culture with target cells. Target cells can be precursor cells and totipotent cells, for example. Feeder cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules (e.g., steroids), nucleic acid molecules, growth factors (e.g., bFGF), other factors (e.g., cytokines such as LIF and steel factor), and metabolic nutrients to target cells. Certain cells, such as immortalized, totipotent cells may not require feeder cells for healthy growth. Feeder cells preferably grow in a mono-layer. Feeder cells can be established from multiple cell types. Examples of these cell types are fetal cells, mouse cells, Buffalo rat liver cells, and oviductal cells. These examples are not meant to be limiting. Tissue samples can be broken down to establish a feeder cell line by methods well known in the art (e.g., by using a blender). Feeder cells may originate from the same or different animal species as the precursor cells. In an example of feeder cells established from fetal cells, ungulate fetuses and preferably bovine fetuses may be utilized to establish a feeder cell line where one or more cell types have been removed from the fetus (e.g., primordial germ cells, cells in the head region, and cells in the body cavity region). When an entire fetus is utilized to establish a fetal feeder cell line, feeder cells (e.g., fibroblast cells) and precursor cells (e.g., primordial germ cells) can arise from the same source (e.g., one fetus). The term “drug” as used herein refers to any type of molecule that retards the normal growth rate of a cell. A normal cell growth rate is measured in the absence of drug. A drug may also lyse cells. In another aspect, the invention features a method for producing transgenic ungulates by introducing a large heterologous nucleic acid construct into a nuclear donor cell, then fusing this nuclear donor cell into an enucleated recipient cell to form a nuclear transfer embryo, activating this embryo, and finally transferring this embryo into a maternal host to produce a transgenic animal. In particularly preferred embodiments, the transgenic animal is an ungulate. Nuclear Transfer Most preferably, transgenic animals are prepared by introducing a heterologous nucleic acid molecule, preferably an artificial chromosome, into a nuclear donor cell, then fusing this nuclear donor cell into an enucleated recipient cell, most preferably an enucleated oocyte, to form a nuclear transfer embryo, activating this embryo, and finally transferring this embryo into a maternal host to produce a transgenic animal. In preferred embodiments, the artificial chromosome(s) is introduced into the cybrid by introduction into the nuclear donor cell prior to the fusion with the enucleated recipient cell or enucleated oocyte. In other preferred embodiments, the artificial chromosome(s) is introduced into the cybrid formed by fusion of the nuclear donor cell with the enucleated recipient cell or enucleated oocyte. In yet other preferred embodiments, the artificial chromosome(s) is introduced into the cybrid simultaneously with the fusion of the nuclear donor cell with the enucleated recipient cell or enucleated oocyte The terms “nuclear transfer” and “nuclear transfer procedure” as used herein refer to introducing a full complement of nuclear DNA from one cell to an enucleated cell. Nuclear transfer methods are well known to a person of ordinary skill in the art. See, U.S. Pat. No. 4,994,384 to Prather et al., entitled “Multiplying Bovine Embryos,” issued on Feb. 19, 1991; U.S. Pat. No. 5,057,420 to Massey, entitled “Bovine Nuclear Transplantation,” issued on Oct. 15, 1991; U.S. Pat. No. 5,994,619, issued on Nov. 30, 1999 to Stice et al., entitled “Production of Chimeric Bovine or Porcine Animals Using Cultured Inner Cell Mass Cells; U.K. Patents Nos. GB 2,318,578 GB 2,331,751, issued on Jan. 19, 2000 to Campbell et al. and Wilmut et al., respectively, entitled “Quiescent Cell Populations For Nuclear Transfer”; U.S. Pat. No. 6,011,197 to Strelchenko et al., entitled “Method of Cloning Bovines Using Reprogrammed Non-Embryonic Bovine Cells,” issued on Jan. 4, 2000; and in U.S. patent application Ser. No. 09/753,323 entitled “Method of Cloning Porcine Animals (attorney docket number 030653.0026.CIP1, filed Dec. 28, 2000), each of which are hereby incorporated by reference in its entirety including all figures, tables and drawings. Nuclear transfer may be accomplished by using oocytes that are not surrounded by a zona pellucida. In a nuclear transfer procedure, a nuclear donor cell, or the nucleus thereof, is introduced into a recipient cell. A recipient cell is preferably an oocyte and is preferably enucleated. However, the invention relates in part to nuclear transfer, where a nucleus of an oocyte is not physically extracted from the oocyte. It is possible to establish a nuclear transfer embryo where nuclear DNA from the donor cell is replicated during cellular divisions. See, e.g., Wagoner et al., 1996, “Functional enucleation of bovine oocytes: effects of centrifugation and ultraviolet light,” Theriogenology 46: 279-284. In addition, nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte. Also, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes. The resulting combination of a nuclear donor cell and a recipient cell can be referred to variously as a “nuclear transfer embryo,” a “hybrid cell,” or a “cybrid.” Furthermore, a nuclear donor may arise from an animal of the same species from which a nuclear recipient is isolated. Alternatively, a nuclear donor may arise from an animal of a different specie from which a nuclear recipient is isolated. For example, a differentiated cell isolated from an ear punch of a water buffalo may be utilized as a nuclear donor and an oocyte isolated from a bovine animal may be utilized as a nuclear acceptor. Thus, xenospecific nuclear transfer is contemplated by the instant invention. The term “nuclear donor” as used herein refers to any cell, or nucleus thereof, having nuclear DNA that can be translocated into an oocyte. A nuclear donor may be a nucleus that has been isolated from a cell. Multiple techniques are available to a person of ordinary skill in the art for isolating a nucleus from a cell and then utilizing the nucleus as a nuclear donor. See, e.g., U.S. Pat. Nos. 4,664,097, 6,011,197, and 6,107,543, each of which is hereby incorporated by reference in its entirety including all figures, tables and drawings. Any type of cell can serve as a nuclear donor. Examples of nuclear donor cells include, but are not limited to, cultured and non-cultured cells isolated from an embryo arising from the union of two gametes in vitro or in vivo; embryonic stem cells (ES cells) arising from cultured embryonic cells (e.g., pre-blastocyst cells and inner cell mass cells); cultured and non-cultured cells arising from inner cell mass cells isolated from embryos; cultured and non-cultured pre-blastocyst cells; cultured and non-cultured fetal cells; cultured and non-cultured adult cells; cultured and non-cultured primordial germ cells; cultured and non-cultured germ cells (e.g., embryonic germ cells); cultured and non-cultured somatic cells isolated from an animal; cultured and non-cultured cumulus cells; cultured and non-cultured amniotic cells; cultured and non-cultured fetal fibroblast cells; cultured and non-cultured genital ridge cells; cultured and non-cultured differentiated cells; cultured and non-cultured cells in a synchronous population; cultured and non-cultured cells in an asynchronous population; cultured and non-cultured serum-starved cells; cultured and non-cultured permanent cells; and cultured and non-cultured totipotent cells. See, e.g., Piedrahita et al., 1998, Biol. Reprod. 58: 1321-1329; Shim et al., 1997, Biol. Reprod. 57: 1089-1095; Tsung et al., 1995, Shih Yen Sheng Wu Hsueh Pao 28: 173-189; and Wheeler, 1994, Reprod. Fertil. Dev. 6: 563-568, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables. In addition, a nuclear donor may be a cell that was previously frozen or cryopreserved. The term “activation” refers to any materials and methods useful for stimulating a cell to divide before, during, and after a nuclear transfer step. Cybrids may require stimulation in order to divide after a nuclear transfer has occurred. The invention pertains to any activation materials and methods known to a person of ordinary skill in the art. Although electrical pulses are sometimes sufficient for stimulating activation of cybrids, other means are sometimes useful or necessary for proper activation of the cybrid. Chemical materials and methods useful for activating embryos are described below in other preferred embodiments of the invention. Examples of non-electrical means for activation include agents such as ethanol; inositol trisphosphate (IP 3 ); Ca ++ ionophores (e.g., ionomycin) and protein kinase inhibitors (e.g., 6-dimethylaminopurine (MAP)); temperature change; protein synthesis inhibitors (e.g., cyclohexamide); phorbol esters such as phorbol 12-myristate 13-acetate (PMA); mechanical techniques; and thapsigargin. The invention includes any activation techniques known in the art. See, e.g., U.S. Pat. No. 5,496,720, entitled “Parthenogenic Oocyte Activation” to Susko-Parrish et al., issued on Mar. 5, 1996; and U.S. Pat. No. 6,077,710, issued on Jun. 20, 2000, each of which is incorporated by reference herein in its entirety, including all figures, tables, and drawings. The term “fusion” as used herein in reference to nuclear transfer refers to the combination of portions of lipid membranes corresponding to the nuclear donor and the recipient oocyte. Lipid membranes can correspond to the plasma membranes of cells or nuclear membranes, for example. The fusion can occur between the nuclear donor and recipient oocyte when they are placed adjacent to one another, or when the nuclear donor is placed in the perivitelline space of the recipient oocyte, for example. Specific examples for translocation of the totipotent mammalian cell into the oocyte are described hereafter in other preferred embodiments. These techniques for translocation are fully described in the references cited previously herein in reference to nuclear transfer. The term “electrical pulses” as used herein refers to subjecting the nuclear donor and recipient oocyte to electric current. For nuclear transfer, the nuclear donor and recipient oocyte can be aligned between electrodes and subjected to electrical current. The electrical current can be alternating current or direct current. The electrical current can be delivered to cells for a variety of different times as one pulse or as multiple pulses. The cells are typically cultured in a suitable medium for the delivery of electrical pulses. Examples of electrical pulse conditions utilized for nuclear transfer are described in the references and patents previously cited herein in reference to nuclear transfer. The term “fusion agent” as used herein in reference to nuclear transfer refers to any compound or biological organism that can increase the probability that portions of plasma membranes from different cells will fuse when a totipotent mammalian cell nuclear donor is placed adjacent to the recipient oocyte. In preferred embodiments fusion agents are selected from the group consisting of polyethylene glycol (PEG), trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses, and Sendai virus. These examples are not meant to be limiting and other fusion agents known in the art are applicable and included herein. The term “suitable concentration” as used herein in reference to fusion agents, refers to any concentration of a fusion agent that affords a measurable amount of fusion. Fusion can be measured by multiple techniques well known to a person of ordinary skill in the art, such as by utilizing a light microscope, dyes, and fluorescent lipids, for example. The term “totipotent” as used herein refers to a cell, embryo, or fetus capable of giving rise to a live born animal. The term “totipotent” can also refer to a cell that gives rise to all of the cells in a particular animal. A totipotent cell can give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps. Totipotent cells, embryos, and fetuses may also be used to generate incomplete animals such as those useful for organ harvesting, e.g., having genetic modifications to eliminate growth of an organ or appendage by manipulation of a homeotic gene. The term “live born” as used herein preferably refers to an animal that exists ex utero. A “live born” animal may be an animal that is alive for at least one second from the time it exits the maternal host. A “live born” animal may not require the circulatory system of an in utero environment for survival. A “live born” animal may be an ambulatory animal. Such animals can include pre- and post-pubertal animals. As discussed previously, a live born animal may lack a portion of what exists in a normal animal of its kind. In preferred embodiments, (1) totipotent cells arise from at least one precursor cell; (2) a precursor cell is isolated from and/or arises from any region of a ungulate animal; (3) a precursor cell is isolated from and/or arises from any cell in culture; (4) a precursor cell is selected from the group consisting of a primary cell, a non-embryonic cell, a non-fetal cell, a differentiated cell, an undifferentiated cell, a somatic cell, an embryonic cell, a fetal cell, an embryonic stem cell, a primordial germ cell, a genital ridge cell, a cumulus cell, an amniotic cell, a fetal fibroblast cell, a uterine cell, an ovarian follicular cell, a cumulus cell, an hepatocyte, an embryonic germ cell, an adult cell, a cell isolated from an asynchronous population of cells, and a cell isolated from a synchronized population of cells where the synchronous population is not arrested in the G 0 stage of the cell cycle; (5) totipotent cells have a morphology of an embryonic germ cell. The terms “precursor cell” or “precursor cells” as used herein refer to a cell or cells used to create a cell line of totipotent cells. Precursor cells can be isolated from, any animal, preferably from a mammal, and more preferably from an ungulate. The precursor cell or cells may be isolated from nearly any cellular entity. For example, a precursor cell or cells may be isolated from blastocysts, embryos, fetuses, and cell lines (e.g., cell lines established from embryonic cells), preferably isolated from fetuses and/or cell lines established from fetal cells, and more preferably isolated from ex utero animals and/or cell cultures and/or cell lines established from such ex utero animals. An ex utero animal may exist as a newborn animal, adolescent animal, yearling animal, and adult animal. The ex utero animals may be alive or post mortem. Examples of precursor cells include, but are not limited to, non-embryonic cells; non-fetal cells; differentiated cells; adult cells; somatic cells; embryonic cells; fetal cells; embryonic stem cells; primordial germ cells; genital ridge cells; uterine cells; amniotic cells; ovarian follicular cells; cumulus cells; cells isolated from an asynchronous population of cells; and cells isolated from a synchronized population of cells where the synchronous population is not arrested in the G 0 stage of the cell cycle; and any of the forgoing that are cultured, cultured as cell lines and/or totipotent. The term “arises from” as used herein refers to the conversion of one or more cells into one or more cells having at least one differing characteristic. For example, (1) a non-totipotent precursor cell can be converted into a totipotent cell by utilizing features of the invention described hereafter; (2) a precursor cell can develop a cell morphology of an embryonic germ cell; (3) a precursor cell can give rise to a cultured cell; (4) a precursor cell can give rise to a cultured cell line; and (5) a precursor cell can give rise to a cultured permanent cell line. A conversion process can be referred to as a reprogramming step. In addition, the term “arises from” refers to establishing totipotent embryos from totipotent cells of the invention by using a nuclear transfer process, as described hereafter. The terms “reprogramming” or “reprogrammed” as used herein refer to materials and methods that can convert a non-totipotent cell into a totipotent cell. Distinguishing features between totipotent and non-totipotent cells are described previously. An example of materials and methods for converting non-totipotent cells into totipotent cells is to incubate precursor cells with a receptor ligand cocktail. Receptor ligand cocktails are described hereafter. In preferred embodiments, culturing of a cell is a sufficient stimulus to render a cell totipotent. The term “reprogramming” or “reprogrammed” as used herein can also refer to materials and methods that can convert a cell into another cell having at least one differing characteristic. Also, such materials and methods may reprogram or convert a cell into another cell type that is not typically expressed during the life cycle of the former cell. For example, (1) a non-totipotent cell can be reprogrammed into an totipotent cell; (2) a precursor cell can be reprogrammed into a cell having a morphology of an EG cell; and (3) a precursor cell can be reprogrammed into a totipotent cell. An example of materials and methods for converting a precursor cell into a totipotent cell having EG cell morphology is described hereafter. The term “isolated” as used herein in reference to cells refers to a cell that is mechanically separated from another group of cells. Examples of a group of cells are a developing cell mass, a cell culture, a cell line, and an animal. These examples are not meant to be limiting and the invention relates to any group of cells. Methods for isolating one or more cells from another group of cells are well known in the art. See, e.g., Culture of Animal Cells: a manual of basic techniques (3 rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan, John Wiley and Sons, Ltd. The terms “cryopreservation” or “cryopreserved” as used herein refer to freezing a cell, embryo, or animal of the invention. The cells, embryos, or portions of animals of the invention are frozen at temperatures lower than 0° C., preferably lower than −80° C., more preferably at temperatures lower than −140° C., and most preferably at temperatures lower than −196° C. Cells and embryos in the invention can be cryopreserved for an indefinite amount of time. It is known that biological materials can be cryopreserved for more than fifty years. For example, semen that is cryopreserved for more than fifty years can be utilized to artificially inseminate a female bovine animal. Methods and tools for cryopreservation are well-known to those skilled in the art. See, e.g., U.S. Pat. No. 5,160,312, entitled “Cryopreservation Process for Direct Transfer of Embryos,” issued to Voelkel on Nov. 3, 1992, hereby incorporated by reference herein in its entirety, including all figures, tables, and drawings. For the purposes of the present invention, the terms “embryo” or “embryonic” as used herein refer to a developing cell mass that has not implanted into the uterine membrane of a maternal host. Hence, the term “embryo” as used herein can refer to a fertilized oocyte, a cybrid (defined herein), a pre-blastocyst stage developing cell mass, and/or any other developing cell mass that is at a stage of development prior to implantation into the uterine membrane of a maternal host. Embryos of the invention may not display a genital ridge. Hence, an “embryonic cell” is isolated from and/or has arisen from an embryo. An embryo can represent multiple stages of cell development. For example, a one cell embryo can be referred to as a zygote, a solid spherical mass of cells resulting from a cleaved embryo can be referred to as a morula, and an embryo having a blastocoel can be referred to as a blastocyst. The terms “enucleated oocyte” or “enucleated recipient cell” as used herein refer to an oocyte which has had its nucleus removed. Typically, a needle can be placed into an oocyte and the nucleus can be aspirated into the inner space of the needle. The needle can be removed from the oocyte without rupturing the plasma membrane. This enucleation technique is well known to a person of ordinary skill in the art. See, U.S. Pat. No. 4,994,384; U.S. Pat. No. 5,057,420; and Willadsen, 1986, Nature 320:63-65. An enucleated oocyte can be prepared from a young or an aged oocyte. An enucleated oocyte is preferably prepared from an oocyte that has been matured, in vitro or in vivo, for some period of time. This time can vary, depending on the source species for the oocyte. For example, bovine oocytes are preferably matured for between 10 hours and 40 hours, more preferably for between 16 hours and 36 hours, and most preferably between 20 hours and 32 hours. In contrast, porcine oocytes are preferably matured for greater than 24 hours, and more preferably matured for greater than 36 hours. In particularly preferred embodiments, a porcine oocyte is matured for more than 40 hours, up to about 96 hours, more preferably from 42-54 hours, and even more preferably from 42 to 48 hours. The terms “maturation” and “matured” as used herein refer to process in which an oocyte is incubated in a medium in vitro. Oocytes can be incubated with multiple media well known to a person of ordinary skill in the art. See, e.g., Saito et al., 1992, Roux 's Arch. Dev. Biol. 201: 134-141 for bovine organisms and Wells et al., 1997, Biol. Repr. 57: 385-393 for ovine organisms and also Mattioli et al., 1989, Theriogenology 31: 1201-1207; Jolliff & Prather, 1997, Biol. Reprod. 56: 544-548; Funahashi & Day, 1993, J. Reprod. Fert. 98: 179-185; Nagashima et al., 1997, Mol. Reprod. Dev. 38: 339-343; Abeydeera et al., 1998, Biol. Reprod. 58: 213-218; Funahashi et al., 1997, Biol. Reprod. 57: 49-53; and Sawai et al., 1997, Biol. Reprod. 57: 1-6, each of which are incorporated herein by reference in their entireties including all figures, tables, and drawings. Maturation media can comprise multiple types of components, including microtubule inhibitors (e.g. cytochalasin B), hormones and growth factors. Other examples of components that can be incorporated into maturation media are discussed in WO 97/07668, entitled “Unactivated Oocytes as Cytoplast Recipients for Nuclear Transfer,” Campbell & Wilmut, published on Mar. 6, 1997, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. The time of maturation can be determined from the time that an oocyte is placed in a maturation medium to the time that the oocyte is subject to a manipulation (e.g., enucleation, nuclear transfer, fusion, and/or activation). Oocytes can be matured for any period of time: an oocyte can be matured for greater than 10 hours, greater than 20 hours, greater than 24 hours, greater than 36 hours, greater than 48 hours, greater than 60 hours, greater than 72 hours, and greater than 90 hours. The term “about” with respect to oocyte maturation refers to plus or minus 3 hours. An oocyte can also be matured in vivo. Time of maturation may be the time that an oocyte receives an appropriate stimulus to resume meiosis to the time that the oocyte is manipulated. Similar maturation periods described above for in vitro matured oocytes apply to in vivo matured oocytes. Nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte. In addition, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes. The term “young oocyte” as used herein refers to an oocyte that has been matured in vitro for a time less than or equal to the length of time between the onset of estrus and ovulation in vivo. For example, the onset of estrus is signaled by a surge in leutenizing hormone. A cow typically ovulates about 26 hours following the onset of estrus. Thus, a young oocyte is an oocyte matured for about 26 hours or less, preferably 16 to 17 hours. Methods for measuring the length of time between the onset of estrus and ovulation are well known to the skilled artisan. See, e.g., P. T. Cupps, “Reproduction in Domestic Animals,” Fourth Edition, Academic Press, San Diego, Calif., USA, 1991. For horses, ovulation occurs about 33 hours after onset of estrus; for pigs, about 40 hours; for sheep and goats, about 24-36 hours; for dogs, about 40-50 hours; and for cats, about 24-36 hours. The term “young oocyte” may also refer to an oocyte that has been matured and ovulated in vivo and that is collected at about the time of ovulation. The term about in this context refers to +/−1 hour. Oocytes can be isolated from live animals using methods well known to a person of ordinary skill in the art. See, e.g., Pieterse et al., 1988, “Aspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries,” Theriogenology 30: 751-762. Oocytes can be isolated from ovaries or oviducts of deceased or live born animals. Suitable media for in vitro culture of oocytes are well known to a person of ordinary skill in the art. See, e.g., U.S. Pat. No. 5,057,420, which is incorporated by reference herein. Some young oocytes can be identified by the appearance of their ooplasm. Because certain cellular material (e.g., lipids) have not yet dispersed within the ooplasm. Young oocytes can have a pycnotic appearance. A pycnotic appearance can be characterized as clumping of cytoplasmic material. For example, in bovines, a “pycnotic” appearance is to be contrasted with the appearance of oocytes that are older than 28 hours, which have a more homogenous appearing ooplasm. The term “aged oocyte” as used herein refers to an oocyte that has been matured in vitro for a time greater than the length of time between the onset of estrus and ovulation in vivo. The term “aged oocyte” may also refer to an oocyte that has been matured and ovulated in vivo and that is collected later than about 1 hour after the time of ovulation. An aged oocyte can be identified by its characteristically homogenous ooplasm. This appearance is to be contrasted with the pycnotic appearance of young oocytes as described previously herein. The age of the oocyte can be defined by the time that has elapsed between the time that the oocyte is placed in a suitable maturation medium and the time that the oocyte is activated. The age of the oocyte can dramatically enhance the efficiency of nuclear transfer. For example, an aged oocyte can be more susceptible to activation stimuli than a young oocyte. The term “ovulated in vivo” as used herein refers to an oocyte that is isolated from an animal a certain number of hours after the animal exhibits characteristics that it is in estrus. The characteristics of an animal in estrus are well known to a person of ordinary skill in the art, as described in references disclosed herein. The terms “maternal recipient” and “recipient female” as used herein refer to a female animal which is implanted with an embryo for development of the embryo. A maternal recipient may be either homospecific or xenospecific to the implanted embryo. For example it has been shown in the art that bovine embryos can develop in the oviducts of sheep. Stice & Keefer, 1993, “Multiple generational bovine embryo cloning,” Biology of Reproduction 48: 715-719. Implanting techniques are well known to a person of ordinary skill in the art. See, e.g., Polge & Day, 1982, “Embryo transplantation and preservation,” Control of Pig Reproduction , D J A Cole and G R Foxcroft, eds., London, UK, Butterworths, pp. 227-291; Gordon, 1997, “Embryo transfer and associated techniques in pigs,” Controlled reproduction in pigs (Gordon, ed), CAB International, Wallingford UK, pp 164-182; and Kojima, 1998, “Embryo transfer,” Manual of pig embryo transfer procedures , National Livestock Breeding Center, Japanese Society for Development of Swine Technology, pp 76-79, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. The term “replication unit” as used herein refers to that portion of a chromosome or other DNA molecule capable of being replicated that is copied from a given origin of replication. A chromosome in eukaryotes has many replication units. The term “origin of replication” refers to the location in a DNA molecule where its replication begins. The term “essentially no homologous DNA” means that the DNA molecule in question comprises almost entirely heterologous DNA. Preferably, a molecule which contains essentially no homologous DNA comprises at least 98%, 99%, 99.5%, or 99.9% heterologous DNA when the number of base pairs of heterologous DNA in the molecule is divided by the overall number of base pairs in the molecule. The term “homologous DNA” as used herein refers to DNA having the same nucleic acid sequence as DNA sequences present in cell nuclear DNA. The term “germ line” refers to those cells which give rise to the reproductive cells of an organism. These cells contain the complete haploid genome of an organism and will pass these DNA molecules to the descendants of the organism in question. The term “somatic cell” refers to those cells of an organism which are not involved in the production of gametes, e.g., they are not involved in passing the genome to the next generation of the organism in question. Transgenic Embryos, Fetuses, and Animals In yet another aspect, the instant invention relates in part to any embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention, where one or more cells in these developing cell masses comprise at least one large heterologous nucleic acid construct, most preferably an artificial chromosome. In preferred embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells of the embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention comprise at least one large heterologous nucleic acid construct. Most preferably, between 90% and all of the cells of the embryos, fetuses, and animals emanating from totipotent mammalian cells of the invention comprise at least one large heterologous nucleic acid construct. Such embryos, fetuses, and animals are known in the art as being “transgenic.” In certain embodiments, the large heterologous nucleic acid construct is an artificial chromosome, most preferably an ACEs or a euchromatin-based minichromosome. The cells of the embryos, fetuses, and animals that comprise at least one artificial chromosome preferably comprise ten or fewer artificial chromosomes; more preferably comprise six or fewer artificial chromosomes, four or fewer artificial chromosomes, or two or fewer artificial chromosomes; and most preferably comprise one artificial chromosome. If the cells of the embryos, fetuses, and animals of the invention comprise more than one artificial chromosome, the artificial chromosomes may be identical or may differ from one another. The term “transgenic” as used herein in reference to embryos, fetuses and animals refers to an embryo, fetus or animal comprising one or more cells that contain heterologous nucleic acids. In preferred embodiments, a transgenic embryo, fetus, or animal comprises one or more transgenic cells. While germ line transmission is not a requirement of transgenic embryos, fetuses, or animals as that term is used herein, in particularly preferred embodiments a transgenic embryo, fetus, or animal can pass its transgenic characteristic(s) through the germ line. In certain embodiments, a transgenic embryo, fetus or animal expresses one or more transgenes as transgenic RNA and protein molecules. Most preferably, a transgenic embryo, fetus or animal results from a nuclear transfer procedure using a transgenic nuclear donor cell. Transgenic totipotent mammalian embryos can be established from cultured cybrids emanating from one or more nuclear transfer procedures, where one of the nuclear transfer procedures utilizes a totipotent mammalian cell harboring at least one artificial chromosome as a nuclear donor. A transgenic totipotent fetus can be established, for example, from a transgenic totipotent embryo that has been implanted into the uterus of a suitable female host. Cloned transgenic mammalian animals of the invention can be established from totipotent mammalian cells, totipotent mammalian embryos, and totipotent mammalian fetuses of the invention. In certain embodiments, a transgenic animal embryo is produced by nuclear transfer of a nuclear donor cell into an enucleated recipient cell according to the following method: (a) a heterologous DNA molecule of greater than 100 kilobase pairs is introduced into one or more ungulate cells by microcell fusion; (b) the one or more cells are cultured to provide a cell culture; (c) a nuclear donor cell obtained from the cell culture is fused with an enucleated recipient cell to form a nuclear transfer embryo comprising the heterologous DNA molecule; and (d) the nuclear transfer embryo is activated to provide the transgenic ungulate embryo. In particularly preferred embodiments, method further comprises one or more of the following: the culturing step comprises selection for one or more markers of said heterologous DNA molecule, whereby at least 90% of cells in said cell culture comprise the heterologous DNA molecule; the transgenic animal is an ungulate selected from the group consisting of a bovine, an ovine, a caprine, and a porcine; the heterologous DNA molecule comprises one or more telomeres, one or more centromeres, and one or more origins of replication; the heterologous DNA molecule is contained within the cells of the transgenic ungulate embryo on a replication unit that comprises essentially no homologous DNA; the activated nuclear transfer embryo is cultured to at least the two cell stage, wherein at least 50% of the cells of the transgenic ungulate embryo comprise the heterologous DNA molecule; the nuclear donor cell is selected from the group consisting of a somatic cell, a primordial germ cell, an embryonic germ cell, and an embryonic stem cell; the heterologous DNA molecule comprises a plurality of copies of at least one transgene; the heterologous DNA molecule is between 100 kilobase pairs and 500 megabase pairs in size; and the heterologous DNA molecule is an artificial chromosome. In yet another aspect, the invention features a method of using a cloned transgenic fetus or animal, where one or more cells of the fetus or animal comprise one or more large heterologous nucleic acid constructs. The method of using a cloned transgenic fetus or animal comprises the step of isolating at least one component from the fetus or animal. The term “component” as used herein can relate to any portion of a fetus or animal. A component can be selected from the group consisting of fluid, biological fluid, cell, tissue, organ, gamete, embryo, and fetus. The term “gamete” as used herein refers to any cell participating, directly or indirectly, in the reproductive system of an animal. Examples of gametes are spermatocytes, spermatogonia, oocytes, and oogonia. Gametes can be present in fluids, tissues, and organs collected from animals (e.g., sperm is present in semen). For example, methods of collecting semen for the purposes of artificial insemination are well known to a person of ordinary skill in the art. See, e.g., Physiology of Reproduction and Artificial Insemination of Cattle (2nd edition), Salisbury et al., copyright 1961, 1978, WH Freeman & Co., San Francisco. However, the invention relates to the collection of any type of gamete from an animal. The term “tissue” is defined previously. The term “organ” relates to any organ isolated from a fetus or animal, or any portion of an organ. Examples of organs and tissues are neuronal tissue, brain tissue, spleen, heart, lung, gallbladder, pancreas, testis, ovary, intestine, skin, and kidney. These examples are not limiting and the invention relates to any organ and any tissue isolated from a cloned animal of the invention. In preferred embodiments, (1) fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses can be subject to manipulation; (2) the manipulation comprises isolating at least one component from an animal or fetus; (3) the manipulation comprises the step of cryopreserving the components; (4) the manipulation comprises the step of thawing components; (5) the manipulation comprises the step of separating the semen into X-chromosome bearing semen and Y-chromosome bearing semen; (6) the manipulation comprises methods of preparing the semen for artificial insemination; (7) the manipulation comprises the step of purification of desired polypeptide(s) from the component; (8) the manipulation comprises concentration of the components; and (9) the manipulation comprises the step of transferring one or more cloned cells, cloned tissues, cloned organs, and/or portions of cloned organs to a recipient organism (e.g., the recipient organism may be of a different species than the donor source). The term “separating” as used herein in reference to separating semen refers to methods well known to a person skilled in the art for fractionating a semen sample into sex-specific fractions. This type of separation can be accomplished by using flow cytometers that are commercially available. Methods of utilizing flow cytometers from separating sperm by genetic content are well known in the art. In addition, semen can be separated by its sex-associated characteristics by other methods well known to a person of ordinary skill in the art. See, U.S. Pat. Nos. 5,439,362, 5,346,990, and 5,021,244, entitled “Sex-Associated Membrane Proteins and Methods for Increasing the Probability that Offspring Will Be of a Desired Sex,” Spaulding, issued on Aug. 8, 1995, Sep. 13, 1994, and Jun. 4, 1991 respectively, all of which are incorporated herein by reference in their entireties including all figures, tables, and drawings. Semen preparation methods are well known to someone of ordinary skill in the art. Examples of these preparative steps are described in Physiology of Reproduction and Artificial Insemination of Cattle (2nd. edition), Salisbury et al., copyright 1961, 1978, W.H. Freeman & Co., San Francisco. The term “purification” as used herein refers to increasing the specific activity of a particular polypeptide or polypeptides in a sample. Specific activity can be expressed as the ratio between the activity of the target polypeptide and the concentration of total polypeptide in the sample. Activity can be catalytic activity and/or binding activity, for example. Alternatively, specific activity can be expressed as the ratio between the concentration of the target polypeptide and the concentration of total polypeptide. Purification methods include dialysis, centrifugation, and column chromatography techniques, which are well-known procedures to a person of ordinary skill in the art. See, e.g., Young et al., 1997, “Production of biopharmaceutical proteins in the milk of transgenic dairy animals,” BioPharm 10(6): 34-38. The term “transferring” as used herein can relate to shifting cells, tissues, organs, and/or portions of organs to an animal. The cells, tissues, organs, and/or portions of organs can be, for example, (a) developed in vitro and then transferred to an animal, (b) removed from an animal and transferred to another animal of a different specie, (c) removed from an animal and transferred to another animal of the same specie, (d) removed from one portion of an animal (e.g., the leg of an animal) and then transferred to another portion of the same animal (e.g., the brain of the animal), and/or (e) any combination of the foregoing. The term “transferring” can relate to adding cells, tissues, and/or organs to an animal and can also relate to removing cells, tissues, and/or organs from an animal and replacing them with cells, tissues, and/or organs from another source. The term “transferring” as used herein can also refer to implanting one or more cells, tissues, organs, and/or portions of organs from the cloned mammalian animal into another organism. For example, neuronal tissue from a cloned mammalian organism can be grafted into an appropriate area in the human nervous system to treat neurological diseases such as Alzheimer's disease. Alternatively, cloned cells, tissues, and/or organs originating from a porcine organism may be transferred to a human recipient. Surgical methods for accomplishing this preferred aspect of the invention are well known to a person of ordinary skill in the art. Transferring procedures may include the step of removing or deleting cells, tissues, or organs from a recipient organism before a transfer step. Of particular interest are transgenic animals that express genes that confer resistance or reduce susceptibility to disease. Since multiple genes can be introduced on an ACEs, a series of genes encoding an antigen can be introduced, which upon expression will serve to immunize [in a manner similar to a multivalent vaccine] the host animal against the diseases for which exposure to the antigens provide immunity or some protection. Also of interest are transgenic animals that serve as models of certain diseases and disorders for use in studying the disease and developing therapeutic treatments and cures thereof. Such animal models of disease express genes [typically carrying a disease-associated mutation], which are introduced into the animal on a MAC, preferably an ACEs, and which induce the disease or disorder in the animal. Similarly, MACs carrying genes encoding antisense RNA may be introduced into animal cells to generate conditional “knock-out” transgenic animals. In such animals, expression of the antisense RNA results in decreased or complete elimination of the products of genes corresponding to the antisense RNA. Of further interest are transgenic mammals that harbor MAC-carried genes encoding therapeutic proteins that are expressed in the animal's milk. Transgenic animals for use in xenotransplantation, which express MAC-carried genes that serve to humanize the animal's organs, are also of interest. Genes that might be used in humanizing animal organs include those encoding human surface antigens. The invention relates in part to any disease or parasitic condition known in the art. See, e.g., Hagan & Bruners Infectious Diesases of Domestic Animals (7th edition), Gillespie & Timoney, copyright 1981, Cornell University Press, Ithaca N.Y. Examples of parasites include, but are not limited to, worms, insects, invertebrate, bacterial, viral, and eukaryotic parasites. These parasites can lead to diseased states that can be controlled by the materials and methods of the invention. The term “regulatory element” as used herein refers to a DNA or RNA sequence that can increase or decrease the amount of product produced from another DNA or RNA sequence. The regulatory element can cause the constitutive production of the product (e.g., the product can be expressed constantly). Alternatively, the regulatory element can enhance or diminish the production of a recombinant product in an inducible fashion (e.g., the product can be expressed in response to a specific signal). The regulatory element can be controlled, for example, by nutrition, by light, or by adding a substance to the transgenic organism's system. Examples of regulatory elements well-known to those of ordinary skill in the art are promoters, enhancers, insulators, and repressors. See, e.g., Transgenic Animals, Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood Academic Publishers, Australia, hereby incorporated herein by reference in its entirety including all figures, tables, and drawings. The terms “promoters,” “promoter,” or “promoter elements” as used herein refer to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product. A promoter is preferably operatively linked to the adjacent DNA sequence. A promoter typically increases the amount of recombinant product expressed from a DNA sequence as compared to the amount of the expressed recombinant product when no promoter exists. A promoter from one organism can be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism. In addition, one promoter element can increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem. Hence, one promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well-known to persons of ordinary skill in the art. Examples of promoter elements are described hereafter. The terms “enhancers,” “enhancer” or “enhancer elements” as used herein refer to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer elements are typically located upstream of a promoter element or can be located downstream of the coding DNA sequence (e.g., the DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream of the DNA sequence that encodes the recombinant product. Enhancer elements can increase the amount of recombinant product expressed from a DNA sequence above the increased expression afforded by a promoter element. Multiple enhancer elements are readily available to persons of ordinary skill in the art. The terms “insulators,” “insulator,” or “insulator elements” as used herein refer to DNA sequences that flank the DNA sequence encoding the recombinant product. Insulator elements can direct the recombinant product expression to specific tissues in an organism. Multiple insulator elements are well known to persons of ordinary skill in the art. See, e.g., Geyer, 1997, Curr. Opin. Genet. Dev. 7: 242-248, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. The terms “repressor” or “repressor element” as used herein refer to a DNA sequence located in proximity to the DNA sequence that encodes the recombinant product, where the repressor sequence can decrease the amount of recombinant product expressed from that DNA sequence. Repressor elements can be controlled by the binding of a specific molecule or specific molecules to the repressor element DNA sequence. These molecules can either activate or deactivate the repressor element. Multiple repressor elements are available to a person of ordinary skill in the art. The terms “milk protein promoter,” “urine protein promoter,” “blood protein promoter,” “tear duct protein promoter,” “synovial protein promoter,” “spermatogenesis protein promoter,” and “mandibular gland protein promoter” refer to promoter elements that regulate the specific expression of proteins within the specified fluid or gland or cell type in an animal. For example, a milk protein promoter is a regulatory element that can control the expression of a protein that is expressed in the milk of an animal. Other promoters, such as β-casein promoter, melanocortin promoter, milk serum protein promoter, casein promoter, α-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and α-actin promoter, for example, are well known to a person of ordinary skill in the art. The terms “insertion” and “introduction” as used herein in reference to artificial chromosomes or other large heterologous nucleic acid constructs refer to translocating one or more such artificial chromosomes or constructs from the outside of a cell to the inside of a cell. Insertion can be effected in at least two manners: by mechanical delivery and non-mechanical delivery. The term “mechanical delivery” as used herein refers to processes that utilize an apparatus that directly or indirectly introduces DNA (e.g., one or more artificial chromosomes) into one or more cells. Examples of mechanical delivery of DNA into cells include, but are not limited to, microinjection, particle bombardment, sonoporation, and electroporation. The term “non-mechanical delivery” as used herein refers to non-mechanical processes such as diffusive processes, for example. For instance, non-mechanical delivery may be effected by introducing DNA (e.g., an artificial chromosome) and one or more reagents to a medium bathing cell surfaces, where the reagents increase the probability that the DNA enters the cells. Such reagents are well known in the art, such as liposomes, acyl moieties, peptide moieties, saccharide moieties, and/or polyethylene glycol (PEG), for example. Such reagents may be complexed with the target molecule and the reagents may be introduced to cells in vivo and/or ex vivo. These examples are not meant to be limiting and the invention relates in part to any non-mechanical form of insertion. The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, as well as from the claims. detailed-description description="Detailed Description" end="lead"? |
Method and vehicle for pavement surface dressing |
According to the invention, the surface is dressed by synchronous spreading of binder and gravel on the pavement as follows: a) a first layer of hot bitumen (b1) having a temperature greater than 100° C. is spread on the pavement (S); b) a film of hot water (e) is spread on top of said first layer, c) a second layer of hot bitumen (b2) having a temperature greater than 100° C. is spread on top of said film of hot water; d) a layer of gravel e) is spread on top of all of the above. Pavement dressing. |
1. Method for applying a surface coating by the synchronous spreading of bonding agent and chippings onto a roadway, according to which; a) a first layer of hot bitumen (b1), at a temperature above 100° C., is spread onto the roadway (S); b) onto this first layer is deposited a film of hot water (e), c) onto this film of hot water is spread a second layer of hot bitumen (b2), at a temperature above 100° C.; d) over the whole thing is spread a layer of chippings (c). 2. Method according to claim 1, characterised in that bitumen taken to a temperature close to 130° C. and water taken to a temperature close to 100° C. are used. 3. Method according to claim 1 or 2, characterised in that the chippings are spread less than two seconds and, preferably, less than one second after the second layer of bitumen is spread. 4. Method according to one of claims 1 to 3, characterised in that the total spreading time is less than about four seconds. 5. Vehicle for applying a surface coating by synchronous spreading of bonding agent and chippings onto a roadway, with the chassis (41) bearing a hot bitumen tank (6) and a tipper (7) containing chippings (c), this vehicle being fitted with a first boom (1) for dispensing hot bitumen (b1), which in placed in front—considering the forward operational movement (F) of the vehicle (4)—of a chipping spreader device (70), characterised in that the chassis (41) additionally bears a hot water tank (5), the vehicle being fitted with a second boom (3) for dispensing hot bitumen (b2), also placed in front of said chipping spreader device (70), and with a hot water (e) spray boom (2) placed between the two bitumen dispensing booms (1; 3). |
Electrohydraulic brake system and method for operating the same |
The invention relates to an electrohydraulic brake system in which, in a normal operating mode, a pedal cylinder (1) which is coupled to a brake pedal (16) can be acted on either by a high-pressure reservoir (12) or a low-pressure reservoir (13) via an electrovalve (4). Likewise, a brake cylinder (2) which is coupled to a brake device (17) can be coupled either to the high-pressure reservoir (12) or the low-pressure reservoir (13) via an electrovalve (7). If there is a fault, by switching over electrovalves (3, 5, 6), the system is changed over into a safety mode in which the pedal cylinder (1) and the brake cylinder (2) are coupled directly hydraulically. In order to ensure a defined volume of hydraulic fluid in the hydraulic path (13) here, an additional cylinder (8) is provided whose spring-loaded side which expands in the safety mode can carry off excess hydraulic fluid from the brake cylinder (2). In addition, hydraulic fluid can be removed from the low-pressure reservoir (13) via a nonreturn valve (11). |
1. An electrohydraulic brake system, containing a) a pedal cylinder (1) which is coupled to a brake pedal (16), b) a brake cylinder (2) which is coupled to brake devices (17), c) at least one valve (3, 6) which is preferably electrically activated and which, in a safety mode, can bring about a hydraulic path (13) with a direct connection between the pedal cylinder (1) and brake cylinder (2), wherein the brake system has a balancing device (8, 9, 10, 11) with which, in the safety mode, a defined volume of hydraulic fluid can be brought about in the hydraulic path (13). 2. The electrohydraulic brake system as claimed in claim 1, wherein the balancing device contains an additional cylinder (8) with a spring-loaded piston, the working volume, which expands under spring effect, of the cylinder being capable of being coupled to the hydraulic path (13) in the safety mode by means of a preferably electrically activated valve (9). 3. The brake system as claimed in claim 2, wherein, outside the safety mode, the working volume, which is compressed under spring effect, of the additional cylinder (8) can be coupled to a high-pressure reservoir (12) via a preferably electrically activated valve (10). 4. The brake system as claimed in one of claims 1 to 3, wherein the hydraulic path (13) is coupled to a low-pressure reservoir (13) via a nonreturn valve (11). 5. The brake system as claimed in one of claims 1 to 4, wherein the latter has a low-pressure reservoir (13) to which, in the safety mode, the respective second sides of the pedal cylinder (1), brake cylinder (2) and/or additional cylinder (8) are coupled. 6. The brake system as claimed in one of claims 1 to 5, wherein the pedal cylinder (1) and/or the brake cylinder (2) is/are coupled to a preferably electrically activated valve (4, 7) via which, outside the safety mode, the cylinder volumes can be connected optionally to a low-pressure reservoir (13) or to a high-pressure reservoir (12). 7. A method for operating an electrohydraulic brake system, in particular a brake system as claimed in one of claims 1 to 6, containing a) a pedal cylinder (1) which is coupled to a brake pedal (16), b) a brake cylinder (2) which is coupled to brake devices (17), in which case, at the changeover into a safety mode, a hydraulic path (13) is brought about with a direct connection between the pedal cylinder (1) and brake cylinder (2), wherein, in the safety mode, a defined volume of hydraulic fluid is brought about in the hydraulic path (13). 8. The method as claimed in claim 7, wherein, at the changeover into the safety mode, hydraulic fluid is sucked out of the brake cylinder (2) until its piston has reached its home position. 9. The method as claimed in claim 7 or 8, wherein hydraulic fluid is fed to the hydraulic path (13) if the pressure there drops below a predefined pressure. 10. The method as claimed in one of claims 7 to 9, wherein, outside the safety mode, high pressure or low pressure is applied to the pedal cylinder (1) and/or brake cylinder (2) under the control of electrovalves (4, 7). |
Resin impregnated filter media |
The present invention discloses a filter medium and a method of manufacturing a filter medium. The filter medium is created using a fabric substrate of a woven or non woven material. The substrate is impregnated with a thermosetting resin that is subsequently heat cured thereby encapsulating the fibers of the substrate. Further a rheology modifier may be mixed with the resin before impregnation to control the flow properties of the resin. |
1. A process of manufacturing a filter medium, comprising: impregnating a substrate with thermosetting resin said substrate being formed from intertwined yarns; and subsequently curing the thermosetting resin by heating the impregnated substrate to the curing temperature of the resin used, to thereby encapsulate the yarns of the substrate with the resin so that the yarns are mutually bonded where they contact each other. 2. A process according to claim 1 said resin is mixed with a rheology modifier before application to control the viscosity and flow properties of the resin. 3. A process according to claim 2 wherein the rheology modifier comprises below 5% of the resin composition by weight. 4. A process according to claim 2 wherein the rheology modifier comprises hydroxyl-ethyl cellulose. 5. A process according to claim 2 wherein the resin is diluted to below 15% solids content before addition of the rheology modifier. 6. A process according to claim 2 wherein the thermosetting resin is selected from the group consisting of: phenolic, epoxy, formaldehyde, amino furan, melamine, silicone, unsaturated polyether, polyurethane, polyamide, fluorocarbon based thermosetting resin, cross linked thermoplastic based thermosetting resin and mixtures thereof. 7. A process according to claim 1 wherein the substrate is selected from the group consisting of: woven fabric, non-woven fabric, spun bonded yarns, thermo-bonded yarns, and melt blown polymeric yarns. 8. A process according to claim 7 wherein the polymeric yarns are a blend of two or more materials selected from the group consisting of: polyester, polypropylene, polyamide, polyethylene, and polyurethane. 9. A process according to claim 7 wherein the substrate includes a metal or ceramic mesh screen. 10. A process according to claim 1 wherein the coated fabric is subjected to an intermediate heating, before curing to a temperature below the curing temperature to remove water or other solvent. 11. A filter medium comprising a substrate of a fabric impregnated with a thermosetting resin cured by subsequent heating of the impregnated substrate to the curing temperature of the resin. 12. A filter medium according to claim 11 wherein said resin contains a rheology modifier to control the viscosity and flow properties of the resin. 13. A filter medium according to claim 12 wherein the rheology modifier comprises below 5% of the resin composition by weight. 14. A filter medium according to claim 12 wherein the rheology modifier comprises hydroxyl-ethyl cellulose. 15. A filter medium according to claim 12 wherein the resin is diluted to below 15% solids content before addition of the rheology modifier. 16. A filter medium according to of claim 11 wherein the thermosetting resin is selected from the group consisting of: phenolic, epoxy, formaldehyde, amino furan, melamine, silicone, unsaturated polyether, polyurethane, polyamide, fluorocarbon based thermosetting resin, cross linked thermoplastic based thermosetting resin and mixtures thereof. 17. A filter medium according to claim 11 wherein the substrate is selected from the group consisting of: woven fabric, non-woven fabric, spun bonded yarns, thermo-bonded yarns, and melt blown polymeric yarns. 18. A filter medium according to claim 17 wherein the polymeric yarns are a blend of two or more materials selected from the group consisting of: polyester, polypropylene, polyamide, polyethylene, and polyurethane. 19. A filter medium according to claim 17 wherein the substrate includes a metal or ceramic mesh screen. |
Image pickup apparatus and method |
The present invention relates to an image capturing apparatus and method in which the image recording time is reduced and the memory capacity required for compression is also reduced. A number-of-bytes calculation unit 302 determines the number of bytes after compression based on an integrated value of high-frequency integrated data supplied from a high-frequency integration processor. Based on the determined number of bytes, a Q-scale calculation unit 303 determines a Q-scale based on which the image data can be compressed one time to a predetermined data size. A Q-table generation unit 304 generates a Q-table based on the Q-scale. A DCT unit 321 performs a discrete cosine transform on the input image data. A quantization processor 322 adjusts the compression ratio of the image data based on the up-to-date Q-table supplied from the Q-table generation unit 304. A variable-length coding processor 323 encodes the image data with variable length coding such as Huffman coding, and outputs the resulting compressed image data. The present invention is applicable to digital cameras. |
1. An image capturing apparatus having a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data, said image capturing apparatus comprising: high-frequency integration means for integrating a high-frequency component of the obtained image data in the monitoring mode; and compression processing means for compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration of the high-frequency integration means, wherein the high-frequency integration means comprises: extraction means for extracting the high-frequency component of the image data; absolute-value determination means for determining an absolute value of the extracted high-frequency component of the image data; and absolute-value integration means for integrating the absolute value of the high-frequency component of the image data determined by the absolute-value determination means; and the compression processing means comprises: number-of-compressed-bytes calculation means for determining the number of compressed bytes of the photographic image data to be recorded based on the integrated value obtained by integration of the high-frequency integration means; quantization-scale calculation means for determining, based on the number of compressed bytes determined by the number-of-compressed-bytes calculation means, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; quantization-table generation means for generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by the quantization-scale calculation means; and compression means for compressing the photographic image data based on the quantization table generated by the quantization-table generation means. 2. An image capturing apparatus according to claim 1, wherein the compression processing means further compresses thumbnail image data corresponding to a thumbnail image obtained by reducing the size of a photographic image corresponding to the photographic image data; based on the integrated value obtained by integration of the high-frequency integration means, the number-of-compressed-bytes calculation means further determines the number of compressed bytes of the thumbnail image data to be recorded; based on this number of compressed bytes determined by the number-of-compressed-bytes calculation means, the quantization-scale calculation means further determines a quantization scale based on which the thumbnail image data is compressed one time to a predetermined number of bytes; based on the quantization scale determined by the quantization-scale calculation means, the quantization-table generation means further generates a quantization table for use in compression of the thumbnail image data; and based on the quantization table generated by the quantization-table generation means, the compression means further compresses the thumbnail image data. 3. An image capturing apparatus according to claim 1, wherein the number-of-compressed-bytes calculation means determines the number of compressed bytes so as to increase as the integrated value of the high-frequency integration means is higher; and the quantization-scale calculation means determines the quantization scale so as to provide a higher compression ratio as the number of bytes when the photographic image data is compressed is greater. 4. An image capturing apparatus according to claim 1, wherein the high-frequency integration means integrates the high-frequency component of the photographic image data processed by predetermined image signal processing. 5. An image capturing method for an image capturing apparatus having a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data, said image capturing method comprising: a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode; and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step, wherein the high-frequency integration step includes: an extraction step of extracting the high-frequency component of the image data; an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data; and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step; and the compression processing step includes: a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the photographic image data to be recorded based on the integrated value obtained by integration performed in the high-frequency integration step; a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by performing the quantization-scale calculation step; and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step. 6. A recording medium having a computer-readable program for an image capturing apparatus recorded therein, said image capturing apparatus having a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data, the program comprising: a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode; and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step, wherein the high-frequency integration step includes: an extraction step of extracting the high-frequency component of the image data; an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data; and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step; and the compression processing step includes: a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the recorded photographic image data based on the integrated value obtained by integration performed in the high-frequency integration step; a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by performing the quantization-scale calculation step; and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step. 7. A computer-executable program for controlling an image capturing apparatus having a monitoring mode in which image data obtained by capturing an object image is monitored, and a photographic image data recording mode in which image data corresponding to a still image which a user instructs recording of is recorded as photographic image data, the program comprising: a high-frequency integration step of integrating a high-frequency component of the obtained image data in the monitoring mode; and a compression processing step of compressing the photographic image data to be recorded in the photographic image data recording mode based on an integrated value obtained by integration performed in the high-frequency integration step, wherein the high-frequency integration step includes: an extraction step of extracting the high-frequency component of the image data; an absolute-value determination step of determining an absolute value of the extracted high-frequency component of the image data; and an absolute-value integration step of integrating the absolute value of the high-frequency component of the image data determined by performing the absolute-value determination step; and the compression processing step includes: a number-of-compressed-bytes calculation step of determining the number of compressed bytes of the photographic image data to be recorded based on the integrated value obtained by integration performed in the high-frequency integration step; a quantization-scale calculation step of determining, based on the number of compressed bytes determined by performing the number-of-compressed-bytes calculation step, a quantization scale based on which the photographic image data is compressed one time to a predetermined number of bytes; a quantization-table generation step of generating a quantization table for use in compression of the photographic image data based on the quantization scale determined by performing the quantization-scale calculation step; and a compression step of compressing the photographic image data based on the quantization table generated by performing the quantization-table generation step. |
<SOH> BACKGROUND ART <EOH>In digital still cameras and portable information terminal devices having a digital still camera function, such as video cameras and PDAs, image data corresponding to captured photographic images is recorded as digital data. However, since the image data has a large data size and requires a large memory capacity for recording, typically, the image data is compressed according to, for example, a JPEG (Joint Photographic Experts Group) method or the like before it is recorded. FIG. 1 is a block diagram showing an example structure of a JPEG compression unit of the related art for compressing image data according to a JPEG method. In FIG. 1 , image data converted to an image format suitable for compression is input to a JPEG compression unit 1 through an input terminal 11 , and is supplied to a DCT (Discrete Cosine Transform) unit 12 . The DCT unit 12 performs a discrete cosine transform on the supplied image data to convert the image data from the time-domain component to the frequency-domain component, and supplies the resulting data to a quantization processor 13 . The quantization processor 13 adjusts the compression ratio of the image data based on a Q-table formed of a table of quantized coefficients supplied from a fixed-length Q-(quantized coefficient) table generation unit 17 , and supplies the image data to a variable-length coding processor 14 . The variable-length coding processor 14 encodes the image data with variable length coding such as Huffman coding, and the resulting compressed image data is output from an output terminal 19 . The variable-length coding processor 14 is also connected with a number-of-bytes calculation unit 15 , and the compressed image data output from the variable-length coding processor 14 is also supplied to the number-of-bytes calculation unit 15 . The number-of-bytes calculation unit 15 determines the number of bytes of the compressed image data corresponding to one screen, and supplies the result to a Q-scale calculation unit 16 . The Q-scale calculation unit 16 calculates the deviation between the input number of bytes and an expected number of bytes after compressing the photographic image data to determine the amount of adjustment of the compression ratio (Q-scale), and supplies the determined Q-scale to the fixed-length Q-table generation unit 17 . The Q-table generation unit 17 generates a new Q-table based on the Q-scale supplied from the Q-scale calculation unit 16 and a predetermined Q-table supplied from a Q-table unit 18 , and supplies the generated Q-table to the quantization processor 13 . The quantization processor 13 again adjusts the compression ratio of the image data based on the new Q-table supplied from the Q-table generation unit 17 . By repeating the foregoing compression process, the JPEG compression unit 1 can compress the input image data to the predetermined data size. However, the above-described method causes a reduction in image quality more than necessary due to block noise or mosquito noise if the compression ratio is set high so that any type of image data can be compressed one time to a predetermined data size. Therefore, a problem is that, typically, the compression process must be performed, for example, two or three times in order to compress various types of image data using appropriate compression ratios, thus extending the time required for compression of image data. Another problem is that the above-described method requires a memory for storing the original image data to be compressed since the compression process must be repeated, thus increasing the memory capacity required for compression. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a block diagram showing an example structure of a JPEG compression unit of the related art for compressing image data according to a JPEG method. FIG. 2 is a block diagram showing an example of the basic structure of an image capturing apparatus according to the present invention. FIG. 3 is a block diagram showing an example structure of internal components of a high-frequency integration processor shown in FIG. 2 . FIG. 4 is a block diagram showing an example structure of internal components of a JPEG compression processor shown in FIG. 2 . FIG. 5 is a flowchart illustrating a high-frequency integration process. FIG. 6 is a flowchart illustrating a JPEG compression process. detailed-description description="Detailed Description" end="lead"? |
Managing coherence via put/get windows |
A method and apparatus for managing coherence between two processors of a two processor node of a multi-processor computer system. Generally the present invention relates to a software algorithm that simplifies and significantly speeds the management of cache coherence in a message passing parallel computer, and to hardware apparatus that assists this cache coherence algorithm. The software algorithm uses the opening and closing of put/get windows to coordinate the activated required to achieve cache coherence. The hardware apparatus may be an extension to the hardware address decode, that creates, in the physical memory address space of the node, an area of virtual memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements. |
1. A method of simplifying and speeding the management of cache coherence in a message passing parallel supercomputer including two or more non-coherent processor elements, where one processor element is primarily performing calculations, while the other processor element is performing message passing activities, the method comprising the steps: opening and closing a put/get window; performing activities to achieve cache coherence; and using said opening and closing of the put/get window to coordinate the activities to achieve cache coherence. 2. A method according to claim 1, wherein the method is implemented by a software algorithm. 3. A method according to claim 1, wherein said using step includes the step of ensuring that data being sent is not in the cache of either processor, and that the data being received is also not in the cache of either processor. 4. A method according to claim 3, wherein the ensuring step includes the step of loading data into cache by issuing a software command. 5. A program storage device readable by machine, tangibly embodying a program of instructions executable by the machine to perform method steps for simplifying and speeding the management of cache coherence in a message passing parallel supercomputer including two or more non-coherent processor elements, where one processor element is primarily performing calculations, while the other processor element is performing message passing activities, the method steps comprising: opening and closing a put/get window; performing activities to achieve cache coherence; and using said opening and closing of the put/get window to coordinate the activities to achieve cache coherence. 6. A program storage device according to claim 5, wherein said using step includes the step of ensuring that data being sent is not in the cache of either processor, and that the data being received is also not in the cache of either processor. 7. A program storage device according to claim 6, wherein the ensuring step includes the step of loading data into cache by issuing a software command. 8. A system to simplify and speed the management of cache coherence in a message passing parallel supercomputer including two or more non-coherent processor elements, where one processor element is primarily performing calculations, while the other processor element is performing message passing activities, the system comprising: means for opening and closing a put/get window; means for performing activities to achieve cache coherence; and means for using said opening and closing of the put/get window to coordinate the activities to achieve cache coherence. 9. A system according to claim 8, wherein said using means includes means for ensuring that data being sent is not in the cache of either processor, and that the data being received is also not in the cache of either processor. 10. A system according to claim 9, wherein the ensuring means includes means for loading data into cache by issuing a software command. 11. Hardware apparatus to assist achieving cache coherence in a message passing parallel computer including two or more non-coherent processing elements, where one processing element is principally performing calculations, while the second processing element is performing message passing activities, the hardware apparatus comprising: a memory controller to create, in the physical memory address space of the node, an area of virtual memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements. 12. Hardware apparatus according to claim 11, wherein the memory controller allows garbage data, which the processor will never use, to be pulled into the processor's cache, thereby evicting just the modified data and displacing unmodified data with optimal performance. 13. Hardware apparatus according to claim 12, wherein the garbage data does not actually need to be fetched from memory, rather, the memory controller need only instantly reply. 14. Hardware apparatus according to claim 13, wherein only actually modified data is written to memory from cache, while clean data is simply instantly discarded. 15. Hardware apparatus according to claim 14, wherein, when the total size of the put/get window exceeds the size of the processor's cache, cleaning the cache in this manner provides an upper bound on the total amount of work that is required to ensure that no data from the communication area remains in the cache. 16. A method of operating computer hardware apparatus to assist achieving cache coherence in a message passing parallel computer including two or more non-coherent processing elements, where one processing element is principally performing calculations, while the second processing element is performing message passing activities, the method comprising the steps: using a memory controller to create, in the physical memory address space of the node, an area of virtual memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements. 17. A method according to claim 16, wherein the memory controller allows garbage data, which the processor will never use, to be pulled into the processor's cache, thereby evicting just the modified data and displacing unmodified data with optimal performance. 18. A method according to claim 17, wherein the garbage data does not actually need to be fetched from memory, rather, the memory controller need only instantly reply. 19. A method according to claim 18, wherein only actually modified data is written to memory from cache, while clean data is simply instantly discarded. 20. A method according to claim 19, wherein, when the total size of the put/get window exceeds the size of the processor's cache, cleaning the cache in this manner provides an upper bound on the total amount of work that is required to ensure that no data from the communication area remains in the cache. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention This invention relates to the field of distributed-memory message-passing parallel computer design and system software, as applied for example to computation in the field of life sciences. 2. Background Art In provisional patent application No. 60/271,124 titled “A Novel Massively Parallel Supercomputer,” therein is described a massively parallel supercomputer architecture in the form of a three-dimensional torus designed to deliver processing power on the order of teraOPS (trillion operations per second) for a wide range of applications. The architecture comprises 65,536 processing nodes organized as a 64×32×32 three-dimensional torus, with each processing node connected to six (6) neighboring nodes. Each processing node of the supercomputer architecture is a semiconductor device that includes two electronic processors (among other components). One of these processors is designated the “Compute Processor” and, in the common made operation, is dedicated to application computation. The other processor is the “I/O Processor,” which, in the common mode of operation, is a service processor dedicated to performing activities in support of message-passing communication. Each of these processors contains a separate first-level cache (L1) which may contain a copy of data stored in a common memory accessed by both processors. If one processor changes its L1 copy of a memory location, and the other processor has a copy of the same location, the two copies become “coherent” if they are made to be the same. Message passing is a commonly-known form of computer communication wherein processors explicitly copy data from their own memory to that of another node. In the dual-processor node disclosed in the above-identified provisional patent application No. 60/271,124, the I/O Processor is principally used to facilitate message passing between the common memory of a node and the common memory of other nodes. Therefore, it both produces data (when a message is received) that is consumed by the Compute Processor, and consumes data (in order to send a message) that is produced by the Compute Processor. As a result, it is very common for both processors to have a copy of the same memory location in their L1s. If the messages passed are small and many, then the problem is exacerbated. Thus, there is a clear need to find a way to make the L1s of each processor coherent, without extensive circuitry, and with minimal impact on performance. As massively parallel computers are scaled to thousands of processing nodes, typical application messaging traffic involves an increasing number of messages, where each such message contains information communicated by other nodes in the computer. Generally, one node scatters locally-produced messages to some number of other nodes, while receiving some number of remotely produced messages into its local memory. Overall performance for these large-scale computers is often limited by the message-passing performance of the system. For such data transfers, a common message-passing interface, described in the literature (see for example http://www.mpi-forum.org/docs/docs.html, under MPI-2), is known as “one-sided communication.” One-sided communication uses a “put/get” message-passing paradigm, where messages carry the source (for get) or the destination (for put) memory address. In parallel supercomputers operating on a common problem, puts and gets are typically assembled in batches and issued together. This keeps the independently operating processors in rough synchronization, maximizing performance. The time during which puts and gets occur is termed the put/get window. This window extends both in time (when it occurs) and in memory (over the range of memory addresses carried by the put or get messages). FIG. 2 shows a put/get window 30 having a number of distinct messages. Put/get windows extend the concept of coherence to processors on different processing nodes of the massively parallel supercomputer. Implementations of put/get windows must insure that all messages put to a window during the time it is open are received into the memory of the window before the window is closed. Similarly, a get on the memory of the window is only allowed during the time the window is open. Therefore, put/get windows are simply a mechanism for a node to synchronize with remote processors operating on its memory. The management of a put/get window is currently accomplished by either buffering the put/get messages or by using explicit synchronization messages. Buffering the messages consumes memory, which is always in limited supply. Explicit synchronization for each window suffers from the long latency of round-trip messages between all the nodes accessing the window. Therefore, on large-scale machines such as the one described in copending patent application No. ______ (attorney Docket 15275), these approaches do not scale well because of limited memory for buffering, and because the number of nodes accessing any particular window often scales along with the number of processing nodes in the computer. A long-standing problem in the field of computer design, is how to keep these L1 caches coherent. Typical solutions employ techniques known as “snooping” the memory bus of the other processor, which can be slow and reduce the performance of each processor. Alternatively, the processor that contains an old copy in L1 of the data in the common memory, can request a new copy, or mark the old copy obsolete, but this requires knowledge of when the copy became invalid. Sometime this knowledge is incomplete, forcing unnecessary memory operations, further reducing performance. Other computers make use of “interlocks,” whereby one processor is granted permission to use certain data while the other processor cannot, but this permission involves interactions between the two processors, which usually requires additional complex circuitry in the semiconductor device, reducing the performance of the two processors. Still other solutions in common practice disable all caching for areas of memory intended to be shared. This practice penalizes all memory accesses to these areas, not just those to the shared data. |
<SOH> SUMMARY OF THE INVENTION <EOH>An object of this invention is to provide an improved procedure for managing coherence in a parallel processing computer system. Another object of the present invention is to achieve coherency between the first-level caches of the processors of a multi-processor node without extensive circuitry and with minimal impact on the performance of each processor. A further object of the invention is to provide a method and apparatus, working in conjunction with software algorithms, to accomplish efficient high speed message-passing communications between processors or a direct memory access (DMA) device, which maintains coherence without significantly reducing performance. These and other objectives are attained with the method and apparatus of the present invention. In accordance with a first aspect, the invention provides a software algorithm that simplifies and significantly speeds the management of cache coherence in a message passing massively parallel supercomputer (such as the one described in copending patent application No. ______ (attorney Docket 15275)) containing two or more non-coherent processing elements (or even a DMA controller) where one processing element is primarily performing calculations, while the other element is performing message passing activities. In such a massively parallel supercomputer, algorithms often proceed as a series of steps, where each step consists of a computation phase followed by a communication phase. In the communication phase, the nodes exchange data produced by the computation phase and required for the next step of the algorithm. Because of the nature of the algorithms, the phases are usually tightly synchronized, so that the communication happens all at once over the entire machine. Therefore, the cost of managing the synchronization of put/get windows can be amortized over a large number of nodes at the start and end of each communication phase. Briefly, a global operation can be used to open many put/get windows at the start of a communication phase, and a second global operation can be used to close the windows at the end of the communication phase. Because the I/O Processor cannot actually send or receive the messages until after cache coherence has been guaranteed, the invention provides a mechanism to ensure that the data being “put” (sent) is not in the cache of either processor, and that the data being “gotten” (received) is also not in the cache of either processor. By coordinating these activities upon opening and closing the “Put/Get Window”, the invention reduces the total amount of work required to achieve coherence and allow that work to be amortized over a large number of individual messages. Also, since both processing elements within a node must perform this work, the invention enables this to happen concurrently. Further, when required, these activities can be coordinated over a large number of independent nodes in the massively parallel machine by employing the Global Barrier Network described in copending patent application No. ______ (attorney Docket 15275). In accordance with a second aspect, the invention provides a hardware apparatus that assists the above-described cache coherence software algorithm, and limits the total time (or latency) required to achieve cache coherence over the Put/Get Window. This apparatus is a simple extension to the hardware address decoder that creates, in the physical memory address space of the node, an area of memory that (a) does not actually exist, and (b) is therefore able to respond instantly to read and write requests from the processing elements. This further speeds the coherence activities because it allows garbage data (which the processor will never use) to be pulled into the processor's cache, thereby evicting just the modified data and displacing unmodified data with optimal performance. The performance is faster because this garbage data does not actually need to be fetched from memory, rather, the memory controller need only instantly reply. The performance is also faster because only modified data is written to memory from cache, while clean data is simply instantly discarded. Further, for the case where the total size of the “Put/Get Window” exceeds, perhaps greatly, the size of the processor's cache, cleaning the cache in this manner provides an upper bound on the total amount of work that is required to ensure that no data from the communication area remains in the cache. It may be noted that, independent of the above-described software algorithms, this hardware device is useful for computer systems in general which employ a Least Recently Used cache replacement policy. Also, two specific software instructions may be used in the preferred implementation of the invention. One instruction, termed “data cache block flush and invalidate”, may be used to write data from the memory area of the first processor into the shared memory area, while at the same time, preventing the first processor from using data the data written in its memory area. A second software instruction, termed “data cache block zero”, may be used to write data from the memory area of the first processor into the shared memory. By using these, or similar software instructions, the method and apparatus of the invention, working in conjunction with software algorithms, achieve high speed message passing communications between nodes, while maintaining coherence without significantly reducing performance. Further benefits and advantages of the invention will become apparent from a consideration of the following detailed description, given with reference to the accompanying drawings, which specify and show preferred embodiments of the invention. |
Omega aminoalkylamides of R-2 aryl propionic acids as inhibitors of the chemotaxis of polymorphonucleate and mononucleate cells |
(R)-2-Arylpropionamide compounds of formula (I) are described. The process for their preparation and pharmaceutical preparations thereof are also described. The 2-Arylpropionamides of the invention are useful in the prevention and treatment of tissue damage due to the exacerbate recruitment of polymorphonuclear leukocytes (leukocytes PMN) and of monocytes at the inflammatory sites. In particular, the invention relates to the R enantiomers of omega-aminoalkylamides of 2-aryl propionic acids, of formula (I), for use in the inhibition of the chemotaxis of neutrophils and monocytes induced by the C5a fraction of the complement and by other chemotactic proteins whose biological activity is associated with activation of a 7-TD receptor. Selected compounds of formula (I) are dual inhibitors of both the C5a-induced chemotaxis of neutrophils and monocytes and the IL-8-induced chemotaxis of PMN leukocytes. The compounds of the invention are used in the treatment of psoriasis, ulcerative cholitis, glomerular nephritis, acute respiratory insufficiency, idiopathic fibrosis, rheumatoid arthritis and in the prevention and the treatment of injury caused by ischemia and reperfusion. |
1. (R)-2-Aryl-propionamide compounds of formula (I). and pharmaceutically acceptable salts thereof, wherein Ar represents a substituted or non-substituted aryl group; R represents hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, optionally substituted by a CO2R3 group, wherein R3 represents hydrogen or a linear or branched C1-C6 alkyl group or a linear or branched C2-C6 alkenyl group; X represents: linear or branched C1-C6 alkylene, C4-C6 alkenylene, C4-C6 alkynylene, optionally substituted by a CO2R3 group or by a CONHR4 group wherein R4 represents hydrogen, linear or branched C2-C6 alkyl or an OR3 group, R3 being defined as above; a (CH2)m—B—(CH2)n, group, optionally substituted by a CO2R3 or CONHR4 group, as defined above, wherein B is an oxygen or sulfur atom, m is zero or an integer from 2 to 3 and n is an integer from 2 to 3; or B is a CO, SO or CONH group, m is an integer from 1 to 3 and n is an integer from 2 to 3; or X together with the nitrogen atom of the omega-amino group to which it is bound and with the R1 group forms a non-aromatic nitrogen containing 3-7 membered heterocyclic, monocyclic or polycyclic ring wherein the nitrogen atom has a substituent Rc, where Rc represents hydrogen, C1-C4 allyl, C1-C4 hydroxyalkyl, C1-C4 acyl, substituted or non-substituted phenyl, diphenylmethyl; R1 and R2 are independently hydrogen, linear or branched C1-C6 alkyl, optionally interrupted by an O or S atom, a C3-C7 cycloalkyl C3-C6 alkenyl, C3-C6-alkynyl, aryl-C1-C3-alkyl, hydroxy-C2-C3-alkyl group; or R1 and R2 together with the N atom to which they are bound, form a nitrogen containing 3-7 membered heterocyclic ring of formula (II) wherein Y represents a single bond, CH2, O, S, or a N-Rc group as defined above and p represents an integer from 0 to 3; or, R1 being as defined above, R2 represents a group of formula (III): wherein Ra is hydrogen and Rb is hydrogen, hydroxy, C1-C4-alkyl or an NRdRe group wherein Rd and Re are independently hydrogen, C1-C4-alkyl or phenyl; or Ra and Rb, together with the nitrogen atoms to which they are bound, form a 5-7 membered heterocyclic ring, monocyclic or fused with a benzene, pyridine or pyrmidine ring; with the proviso that when Ar is a 4-diphenyl residue and X is an ethylene or propylene residue, R1 and R2 are not ethyl; with the further proviso that, when Ar is a 4-(2-fluoro)diphenyl residue, and X is butylene substituted by a CO2H group, Ra and Rb are not hydrogen, or R1 and R2 are not hydrogen; and with the further proviso that, when Ar is phenyl and X is butylene, R1 and R2 together are not a N-(2-methoxy phenyl) piperazine. 2. (R)-2-Aryl-propionamide compounds of formula (I). and pharmaceutically acceptable salts thereof, wherein Ar represents a substituted or non-substituted aryl group; R represents hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, optionally substituted by a CO2R3 group, wherein R3 represents hydrogen or a linear or branched C1-C6 alkyl group or a linear or branched C2-C6 alkenyl group; X represents: linear or branched C1-C6 alkylene, C4-C6 alkenylene, C4-C6 alkynylene, optionally substituted by a CO2R3 group Or by a CONHR4 group wherein R4 represents hydrogen, linear or branched C2-C6 alkyl or an OR3 group, R3 group, R3 being defined as above; a (CH2)m—B—(CH2)n, group, optionally substituted by a CO2R3 or CONHR4 group, as defined above, wherein B is an oxygen or sulfur atom, m is zero or an integer from 2 to 3 and n is an integer from 2 to 3; or B is a CO, SO or CONH group, m is an integer from 1 to 3 and n is an integer from 2 to 3; or X together with the nitrogen atom of the omega-amino group to which it is bound and with the R1 group forms a non-aromatic nitrogen containing 3-7 membered heterocyclic, monocyclic or polycyclic ring wherein the nitrogen atom has a substituent Rc, where Rc represents hydrogen, C1-C4 alkyl, C1-C4 hydroxylalyl, C1-C4 acyl, substituted or non-substituted phenyl, diphenylmethyl; R1 and R2 are independently hydrogen, linear or branched C1-C6 alkyl optionally interrupted by an O or S atom, a C3-C7 cycloalkyl, C3-C6 alkenyl, C3-C6-alkyl, aryl-C1-C3-alkyl, hydroxy-C2-C3-alkyl group; or R1 and R2 together with the N atom to which they are bound, form a 3-7 membered nitrogen heterocyclic ring of formula (II) wherein Y represents a single bond, CH2, O, S, or a N-Rc group as defined above and p represents an integer from 0 to 3; or, R1 being as defined above, R2 represents a group of formula (III): wherein Ra is hydrogen and Rb is hydrogen, hydroxy, C1-C4-alkyl or an NRdRe group wherein Rd and Re, are each independently, hydrogen, C1-C4-alkyl or phenyl; or Ra and Rb, together with the nitrogen atoms to which they are bound, form a 5-7 membered heterocyclic ring, monocyclic or fused with a benzene, pyridine or pyrmidine ring, with the proviso that Ar is not a dihydropyrrole residue; leukocytes and monocytes. 3. Compounds according to claim 1, wherein Ar is chosen from: a) an Ara, mono- or poly-substituted aryl group of (±)2-aryl-propionic acids selected from alminoprofen, benoxaprofen, carprofen, fenbufen, fenoprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, loxoprofen, R-naproxen, pirprofen and its dehydro and dihydro derivatives, pranoprofen, surprofen, tiaprofenic acid, zaltoprofen; b) an aryl-hydroxymethyl-aryl group of formula (IVa), both as diastereoisomeric mixture and as single S′ and/or R′ 0diastereoisomers wherein, when Ar2 is phenyl Ar1 is selected from the group consisting of phenyl and thien-2-yl while when Ar1 is phenyl, Ar2 is selected from the group consisting of phenyl, 4-thienyl pyridyl; c) an aryl of formula (IVb): Φ-Arb (IVb) wherein Arb is a phenyl mono- and poly-substituted by hydroxy, mercapto, C1-C3-alcoxy, C1-C3-alkylthio, chlorine, fluorine, trifluoromethyl, nitro, amino, C1-C7-acylamino optionally substituted; and Φ is hydrogen; a linear or branched C1-C5 alkyl, C2-C5-alkenyl or C2-C5-alkynyl residue optionally substituted by C1-C3-alkoxycarbonyl, substituted or non-substituted phenyl, 2-, 3- or 4-pyridyl, quinolin-2-yl; a C3-C6-cycloalkyl; 2-furyl; 3-tetrahydrofuryl; 2-thiophenyl; 2-tetrahydrothiophenyl or a residue of formula (IVc) A-(CH2)q- (IVc) wherein A is a C1-C5-dialkylamino group, a C1-C8-(alcanoyl, cycloalcanoyl, arylalcanoyl)-C1-C5-alkylamino group, for example dimethylamino, diethylamino, methyl-N-ethyl-amino, acetyl-N-methyl-amino, pivaloyl-N-ethyl-amino; a nitrogen containing 5-7 membered monocyclic ring optionally containing one or two double bonds and optionally another heteroatom separated by at least 2 carbon atoms from the N atom, so as to form, for example, a 1-pyrrolidino, 2,5-dihydro-pyrrol-1-yl, 1-pyrrol, 1-piperidino, 1-piperazino-4-non-substituted or 4-substituted (methyl, ethyl, 2-hydroxyethyl, benzyl, benzyhydril or phenyl), 4-morpholino, 4-3,5-dimethyl-morpholino, 4-thiomorpholino group; or alternatively, a residue of formula (IVd) wherein Rg is hydrogen, C1-C3-alkyl or the residue of a C1-C3-alcanoic acid; q is zero or the integer 1, d) a 2-(phenylamino)-phenyl of formula (IVe): wherein the substituents P1 and P2 indicate that the two phenyl groups bear, each independently, mono- or poly-substitutions with C1-C4-alkyl, C1-C3-alcoxy groups, chlorine, fluorine and/or trifluoromethyl. 4. Compounds according to any of claims 1 to 3, wherein Ar is chosen from: 4-isobutylphenyl, 4-(2-methyl)allyl-phenyl, 3-phenoxyphenyl, 3-benzoyl-phenyl, 3-acetyl-phenyl, the single diastereoisomers (R), (S) and the diastereoisomeric mixture (R,S) of 3-C6H5—CH(OH)-phenyl, 3-CH3—CH(OH)-phenyl, 5-C6H5-CH(OH)-thienyl, 4-thienyl-CH(OH)-phenyl, 3-(pyrid-3-yl)-CH(OM-phenyl, 5-benzoyl-thien-2-yl, 4 thienoyl-phenyl, 3-nicotinoyl-phenyl, 2-fluoro-4-phenyl, 6-metoxy-2-naphthyl, 5-benzoyl-2-acetoxy-phenyl and 5-benzoyl-2-hydroxy-phenyl. 5. Compounds according to any of claims 1 to 3, wherein Ar is phenyl 3-substituted by a group selected form: isoprop-1-en-1-yl, isopropyl, pent-2-en-3-yl, pent-3-yl, 1-phenylethylen-1-yl, α-methylbenzyl. 6. Compounds according to claim 3, wherein the Ar group of formula IVc is selected from: 4-(pyrrolidin-1-yl)-methyl-phenyl, 3-chloro-4(pyrrolidin-1-yl)-methyl-phenyl, 3-chloro-4-(2,5-dihydro-1-H-pyrrol-1-yl)-methyl-phenyl, 3 chloro-4-(thiomorpholin-4-yl)phenyl, 3-chloro-piperidin-1-yl)-phenyl, 4-((N-ethyl-N-quinolin-2-yl-methylamino)-methyl)phenyl, 3-chloro-4-(morpholin-4-yl)-phenyl. 7. Compounds according to claim 3, wherein the Ar group of formula IVe is selected from: 2-(2,6-dichloro-phenyl-amino)-phenyl, 2-(2,6-dichloro-phenyl-amino)-5-chloro-phenyl, 2-(2,6-dichloro-3-methyl-phenyl-amino)-phenyl, 2-(3-trifluoromethyl-phenyl-amino)-phenyl. 8. Compounds according to any of claims 1 to 7, wherein: R is hydrogen, X is: a linear alkylene optionally substituted at C1 by a —CO2R3 group as defined above; a linear alkylene optionally substituted at C1 by a —CONHR4 group wherein R4 is OH; 2-butynylene, cis-2-butenylene, trans-2-butenylene; 3-oxa-pentylene, 3-thio-pentylene, 3-oxa-hexylene, 3-thio-hexylene; (CH2)m—CO—NH—(CH2)n-wherein m and n are each independently an integer from 2 to 3; (CHR′)—CONH—(CH2)n wherein n is an integer from 2 to 3 and R″ is a methyl, in absolute configuration R or S; or X, together with the N atom of the omega-amino group, forms a nitrogen containing cycloaliphatic ring selected from 1-methyl-piperidin-4-yl and 1,5-tropan-3-yl. 9. Compounds according to any of claims 1 to 8, wherein NR1R2 represents an NH2 group, dimethylamino, diethylamino, diisopropylamino, 1-piperidinyl, 4-morpholyl, 4-thiomorpholyl or, R1 and R2 together form a residue of guanidine, aminoguanidine, hydroxyguanidine, 2-amino-3,4,5,6-tetrahydropyrimidyl, 2-amino-3,5-dihydro-imidazolyl. 10. Compounds according to any of claims 1 to 9, selected from: (R)-2-[(4-isobutyl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(4-dimethylaminobutyl)-propionamide hydrochloride; (R)-2-[(4-isobutyl)phenyl]-N-(3-N-morpholinylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(2-dimethylaminoethyl)propionamide; (R)-2-[(4-isobutyl)phenyl)-propionyl]-N-[2-(4methyl-piperazin-1-yl)ethyl]propionamide; (R)-N-(exo-8-methyl-8-aza-bicyclo[3,2,1]oct-3-yl)-2-[(4-isobutylphenyl)-propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(3-N-thiomorpholinylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[4-(N′-methyl)piperidinyl]propionamide hydrochloride; (R),(S′)-2-[(4-isobutyl)phenyl]-N-(1-carboxy-2-dimethylaminoethyl)-propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-[(1-carboxy-4-piperidin-1-yl)butyl]propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-(1-carboxy-4-aminobutyl)propionamide; (R)-2-(4-isobutyl)phenyl-N-[2-dimethylaminoethyl)aminocarbonylmethyl)]propionamide hydrochloride; 2-(2,6-dichlorophenylamino)-phenyl-N-(3-dimethylaminopropyl)propionamide; (R),(R′,S′)-3-[3-(α-methyl)benzyl]phenyl-N-(3-dimethylaminopropyl)-propionamide; (R-2-[(3-isopropyl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)2-[3-pent-3-yl)phenyl]-N-(3-dimethylaminopropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-(3-guanidylpropyl)propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[(3-hydroxy-guanidyl)propyl]propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[(3-amino-guanidyl)propyl]propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[3-(2-amino-2-imidazoline) propyl]propionamide; (R)-2-[(4-isobutyl)phenyl]-N-[N-methyl-N-(2-hydroxyethyl)aminoethoxy]propionamide; (R),(S′)-2-[(4-isobutyl)phenyl]-N-[1-carboxy-5-aminopentyl]propionamide. 11. (R)2-(4-isobutylphenyl)-N-(3-dimethylaminopropyl)propionamide hydrochloride 12. (R)2-(4-isobutylphenyl)-N-3-(1-piperidinylpropyl)propionamide hydrochloride 13. Compounds according to any of claims 1 to 9 wherein R1 and R2 are groups different from hydrogen. 14. Compounds according to claim 13, wherein X is a linear C2-C4 alkylene. 15. Process for the preparation of (R)-2-aryl propionamide compounds of formula (I) according to claim 1 wherein Ar, X, R, R1 and R2 have the meanings as defined in claim 1, comprising reaction of an activated form of an R-2-arylpropionic acid of formula (V) with an amine of formula (VI) wherein AT is a residue activating the carboxy group of the R-2-arylpropionic acid; with the proviso that Ar is not substituted by a dihydropyrrole residue. 16. Compounds according to claim 1, for use as medicaments. 17. Compounds according to claim 2, for use in the treatment of psoriasis, pemphigus and pemphigoid, rheumatoid arthritis, intestinal chronic inflammatory patologies including ulcerative colitis, acute respiratory distress syndrome, idiopathic fibrosis, cystic fibrosis, chronic obstructive pulmonary disease and glomerulonephritis. 18. Compounds according to claim 2 for use in the prevention and the treatment of injury caused by ischemia and reperfusion. 19. Compounds according to claims 13 and 14, for use as inhibitors of both the C5a-induced chemotaxis of polymorphonucleate leukocytes and monocytes, and the interleukin 8-induced chemotaxis of polymorphonucleate leukocytes. 20. Pharmaceutical compositions containing a compound according to any of claims 1 to 14, in admixture with a suitable carrier thereof. |
<SOH> INTRODUCTION AND BACKGROUND OF THE INVENTION <EOH>Animal studies show that some aminoalkylester and amide prodrugs of racemic ibuprofen and naproxen, in particular some N-(3-diethylaminopropyl)amides, exhibit analgesic and antiinflammatory activity significantly better than the parent compounds, even though “in vitro” they have been found to be poor inhibitors of the synthesis of prostaglandins. All these prodrugs, except a glycine amide, have also been found to be significantly less irritating to the gastric mucosa than their precursor free acids. (Shanbhag V R et al., J. Pharm. Sci., 81, 149, 1992 and references 8-19) therein cited. Piketoprofen [(±)2-(3-benzoylphenyl)-N-(4-methyl-2-pyridinyl)propionamide] and Amtolmetin Guacil (also named guaiacol ester of tolmetinglycinamide, Eufans) are further examples of non steroidal antiinflammatory (NSAI) prodrugs in current therapeutic use. Moderate antiinflammatory activity, minor side effects and good gastro-intestinal tolerance are reported for a series or N-[2-(1-piperidinyl)propyl]amides of some NSAI drugs such as racemic ibuprofen, indomethacin, p-chlorobenzoic acid, acetylsalicyclic acid, diacetylgentisic acid and adamantane-1-carboxylic acid (Nawladonski F. and Reewuski, Pol. J. Chem., 52, 1805, 1978). Other amides of racemic 2-arylpropionic acids have been disclosed by S. Biniecki et al., [PL 114050 (31. 01. 1981)], H. Akguen et al., [Arzneim-Forsch., 46, 891, 1986] and by G. L. Levitt et al., [Russ. J. Org. Chem., 34, 346, 1998]. Anti-inflammatory and analgesic potencies “in vivo”, comparable and sometimes greater than those of the precursor free acids, along with decreased number of gastric lesions, have been reported for some N-3-[(1-piperidinyl)propyl]amides of racemic ketoprofen and flurbiprofen and for certain Mannich bases obtained reacting their amides with formaldehyde and secondary amines such as morpholine, piperidine, dicyclohexylamine, dimethylamine, diethylamine, dibenzylamine and dibutylamine (N. Kawathekar et al., Indian J. Pharm. Sci., 60, 346, 1998). International patent application, WO 00/40088, has recently reported that the mere conversion to an amide derivative of a 2-arylacetic and/or 2-arylpropionic acid is enough to change a selective COX-1 inhibitor into a COX-2 selective inhibitor which explains the decreased gastrolesivity of said amides, for a long time believed to be only NSAI prodrugs. In the past, inhibition of the cyclooxygenase enzymes was known to be proper of the S enantiomer of 2-arylpropionic acids alone, joined together with the portion of R CoA-thioester suffering bioconversion “in vivo”. Therefore, the poor correlation between enzymatic inhibition “in vitro” and analgesic effects “in vivo” found for certain R,S 2-arylpropionic acids (Brune K. et al., Experientia, 47, 257, 1991) has induced to presume that alternative mechanisms, such as inhibition of transcription of the kB-nuclear transcription factor (NF-kB) and/or inhibition of neutrophil chemotaxis induced by interleukin 8 (IL-8), can be operating. R enantiomers of flurbiprofen, ketoprofen, naproxen, thiaprofen and phenoprofen are, in fact, disclosed in WO 00/40088 as inhibitors of the NF-kB transcription factor activation and claimed to be useful in the treatment of NF-kB dependent diseases (asthma, tumor, shock, Crohn'disease and ulcerative colitis, arteriosclerosis, etc). IL-8 is an important mediator of inflammation and has been shown to be a potent chemotactic/cell activator for polymorphonucleate neutrophils and basophils (PMNs), and T lymphocytes. Cellular sources of IL-8 include monocytes, PMNs, endotelial cells, epithelial cells, and keratinocytes when stimulated by factors such as lipopolysaccaride, IL-1 and TNF-α. On the other hand, the complement fragment C5a, in addition to being a direct mediator of inflammation, has been found to induce both IL-8 synthesis and high level of IL-8 release from monocytes. The quantity of IL-8 recovered from C5a activated monocytes in peripheral blood mononuclear cells is up to 1,000 fold greater than that released from comparable numbers of PMNs under similar conditions. Therefore IL-8 released from C5a-activated monocytes may play a significant role in expanding and prolonging cellular infiltration and activation at the sites of infection, inflammation, or tissue injury (Ember J. A. et al., Am. J. Pathol., 144, 393, 1994). In response to immunologic and infective events, activation of the complement system mediates amplification of inflammatory response both via direct membrane action and via release of a series of peptide fragments, generally known as anaphylatoxins, generated by enzymatic cleavage of the C3, C4 and C5 complement fractions. These peptides include C3a, C4a, both made of 77 aminoacids; in turn, C5 convertase cleaves the C5 complement fraction to give the glycoprotein C5a of 74 aminoacids. Anaphilatoxins contribute to the spreading of the inflammatory process by interaction with individual cell components; their common properties are cellular release of vasoactive amines and lysosomal enzymes, contraction of smooth muscle and increased vascular permeability. Moreover, C5a causes chemotaxis and aggregation of neutrophils, stimulates the release of leukotrienes and of oxidized oxygen species, induces the transcription of IL-1 in macrophages and the production of antibodies. The C5a peptide fragment of the complement has been defined as the “complete” pro-inflammatory mediator. On the contrary, other inflammatory mediators such as selected cytokines (IL-8, MCP-1 and RANTES, for example) are highly selective towards self-attracted cells, while histamine and bradykinin are only weak chemotactic agents. Convincing evidences support the involvement of C5a, “in vivo”, in several pathological conditions including ischemia/reperfusion, autoimmune dermatitis, membrane-proliferative idiopathic glomerulonephritis, airway iperresponsiveness and chronic inflammatory diseases, ARDS and COPD, Alzheimer'disease, juvenile rheumatoid arthritis (N. P. Gerard, Ann. Rev. Immunol., 12, 755, 1994). In view of the neuro-inflammatory potential of C5a/C5a-desArg generated by both local complement production and amyloid activation joined with astrocyte and microglia chemotaxis and activation directly induced by C5a, complement inhibitors have been proposed for the treatment of neurological diseases such as Alzheimer'disease (McGeer & McGeer P. L., Drugs, 55, 738, 1998). Therefore, the control of the local synthesis of complement fractions is considered of high therapeutic potential in the treatment of shock and in the prevention of rejection (multiple organ failure and hyperacute graft rejection) (Issekutz A. C. et al., Int. J. Immunopharmacol, 12, 1, 1990; Inagi R. et at., Immunol. Lett., 27, 49, 1991). More recently, inhibition of complement fractions has been reported to be involved in the prevention of native and transplanted kidney injuries taking account of complement involvement in the pathogenesis of both chronic interstitial and acute glomerular renal injuries. (Sheerin N. S. & Sacks S. H., Curr. Opinion Nephrol. Hypert., 7, 395, 1998). Genetic engineering and molecular biology studies led to the cloning of complement receptors (CRs) and to the production of CRs agonists and antagonists. The recombinant soluble receptor CR1 (sCR1), that blocks enzymes activating C3 and C5, has been identified as a potential agent for the suppression of C activation on ischemia/reperfusion injury (Weisman H. F. et al., Science, 239, 146, 1990; Pemberton M. et al., J. Immunol., 150, 5104, 1993). The cyclic peptide F-[OPdChWR], is reported to antagonize the C5a binding to its CD38 receptor on PMNs and to inhibit C5a-dependent chemotaxis and cytokine production by macrophages and rat neutropenia induced by C5a and LPS stimulation (Short A. et al., Br. J. Pharmacol., 126, 551, 1999; Haynes D. R. et al., Biochem. Pharmacol., 60, 729, 2000). Both C5aR antagonist CGS 27913 and its dimer CGS 32359 are reported to inhibit, “in vitro”, C5a binding to neutrophil membranes, intracellular Ca 2+ mobilization, lysozyme release, neutrophil chemotaxis and dermal edema in rabbits (Pellas T. C. et al., J. Immunol., 160, 5616, 1998). Finally, selection from phage libraries with the “phage display” technique has led to the isolation of a specific C5aR antagonist able to decrease inflammatory responses in diseases mediated by immuno-complexes and in ischemia and reperfusion injuries (Heller T. et al., J. Immunol., 163, 985, 1999). Despite their therapeutic potential, only two of the above discussed C5a antagonists have demonstrated activity “in vivo”; furthermore, their use is therapeutically limited by their peptidic nature. (Pellas T. C., Wennogle P., Curr. Pharm. Des., 10, 737, 1999). Characteristic neutrophil accumulation can be observed in some pathologic conditions, for example in the highly inflamed and therapeutically recalcitrant areas of psoriatic lesions. Neutrophils are chemotactically attracted and activated by the sinergistic action of chemokines, IL-8 and Gro-a released by the stimulated keratinocytes, and of the C5a/C5a-desArg fraction produced via the alternative complement pathway activation (T. Terui et al., Exp. Dermatol., 9, 1, 2000). In many circustances it is, therefore, highly desirable to combine inhibition of the chemotaxis induced by C5a and inhibition of the chemotaxis induced by IL-8 in one single agent. Non-peptidic antagonists of complement fractions have also been prepared, for example substituted-4,6-diamino-quinolines. In particular, [N,N″-bis-(4-amino-2-methyl-6-quinolyl)]urea and [6-N-2-chlorocynnamoyl)-4,6-diamino-2-methylquinoline] have been found selective C5R antagonists, their IC 50 ranging between 3.3 and 12 μg/mL (Lanza T. J. et al., J. Med. Chem., 35, 252, 1992). Some serine-protease inhibitors [nafamostat mesilate (FUT 175) and certain analogs] have been recently reported to be inhibitors of both complement activation and C3a/C5a production (Ueda N. et al., Inflammation Res. 49, 42, 2000). U.S. Pat. No. 6,069,172 reports the use of pharmaceutical formulations of R(−)ketoprofen ammonium salts for the inhibition of neutrophil chemotaxis induced by IL-8. WO 00/24710 discloses N-acylsulfonamides of R(−)2-aryl-propionic acids as inhibitors of IL-8 dependent polymorphonucleate leukocytes chemotaxis. Two recent patent applications [WO 01/58852 and WO 01/79189] disclose certain R-2-aryl-propionamides and R-2-(aminophenyl)propionamides useful for preventing leukocyte activation induced by IL-8. We have recently observed that the mere formal reduction of the hetero-aromatic ring of certain R 2-aryl-N-(pyridinyl)propionamides causes marked loss of potency (1 or 2 logarithmic order) in the capacity to inhibit PMN neutrophil chemotaxis induced by IL-8. Unexpectedly, the related R 2-aryl-N-(piperidinyl)propionamides have been found to be potent inhibitors of chemotaxis of human PMN leukocytes and monocytes induced by the C5a fraction of the complement. These unexpected findings have originated a novel family of omega-aminoalkylamides of R-2-aryl-propionic acids which are able to inhibit the chemotactic activity induced by C5a and other chemotactic proteins whose biological activity is associated with activation of a 7-membered-domain receptor (7-TD) homologous to the receptor of C5a (for example, the C3a receptor and the CXCR2 receptor; Neote K. et al., Cell, 72, 415, 1993; Tometta M. A., J. Immunol., 158, 5277, 1997). detailed-description description="Detailed Description" end="lead"? |
<SOH> BRIEF DESCRIPTION OF THE INVENTION <EOH>It is the object of the present invention a novel class of omega-aminoalkylamides of R-2-aryl-propionic acids and pharmaceutical compositions containing them. The position “omega” in the alkyl chain refers to the furthest carbon atom starting from the N atom of the amide group to which said alkyl is linked. Such amides are useful in the inhibition of the chemotactic activation induced by C5a and by other chemotactic proteins whose biological activity is associated with the activation of 7-transmembrane domains (7-TD) receptors homologous to the C5a receptor. In particular such amides are useful in the inhibition of the chemotactic activation of polymorphonucleate leukocytes, monocytes and lymphocytes T induced by the fraction C5a of the complement and in the treatment of pathologies related to said activation. |
Nozzle for injecting sublimable solid particles entrained in gas for cleaning a surface |
Disclosed is a nozzle for injecting sublimable solid particles, which is capable of minimizing consumption of the carrier gas and also maximizing cleaning efficiency. The nozzle comprises a base block having a space in which carrier gas is supplied through a gas supplying pipe; a sub-block having a space in which cleaning medium decompressed by a regulator is supplied through a cleaning medium supplying pipe; a first venturi block having a venturi path for adiabatically expanding the carrier gas supplied from the space of the base block, and a cleaning medium injection path communicating the venturi path and the space of the sub-block and the carrier gas passed through the venturi path; and a second venturi block having a venturi path for adiabatically expanding the mixed gas of the carrier gas and the cleaning medium. |
1. A nozzle for injecting sublimable solid particles entrained in gas for cleaning a surface, comprising: a base block having a space in which carrier gas is supplied through a gas supplying pipe; a sub-block having a space in which cleaning medium decompressed by a regulator is supplied through a cleaning medium supplying pipe; a first venturi block having a venturi path for adiabatically expanding the carrier gas supplied from the space of the base block, and a cleaning medium injection path is adjacent to a throttle portion so as to connect the throttle portion of the venturi path with the space of the sub-block to mix the cleaning medium of the sub-block and the carrier gas passed through the venturi path; and a second venturi block having a venturi path for adiabatically expanding the mixed gas of the carrier gas and the cleaning medium, wherein the venturi path of the first venturi block has an acute angle with respect to the cleaning medium injection path. 2. The nozzle of claim 1, further comprising an intermediate block having a path, which is disposed between the first and second venturi blocks, for promoting the mixture of the carrier gas and the cleaning medium in the mixed gas moving from the path of the first venturi block to the path of the second venturi block and thus inducing growth of snow particles. 3. The nozzle of claim 1, wherein the first and second venturi blocks respectively have a plurality of venturi paths disposed in parallel, and the sub-block has the same number of cleaning injection paths as the number of venturi paths. 4. The nozzle of claim 1, wherein the venturi path of the first venturi block has an angle of 15 to 60° with respect to the cleaning medium injection path. 5. The nozzle of claim 1, wherein the carrier gas supplying pipe has a slit at an end thereof so that the carrier gas is injected at a desired angle range when supplied to the space of the base block. 6. The nozzle of claim 5, wherein the carrier gas injected through the slit has an angle of 116°. 7. The nozzle of claim 1, wherein the cleaning medium supplied to the first venturi path has a pressure of 10 to 50 psi, and the carrier supplied to the space of the base block has a pressure of 60 to 100 psi. 8. The nozzle of claim 1, wherein the cleaning medium is CO2+ or Ar+. 9. The nozzle of claim 1, wherein the carrier gas is N2 gas or clean dry air. |
<SOH> BACKGROUND ART <EOH>In order to clean pollutants such as fine particles on a surface of a wafer, an LCD, a color filter or various glass substrates, there has been proposed various techniques. Particularly, in the semiconductor industry, high-pressure liquid is used independently or used in a state of being combined with brushes to remove the polluted fine particles from a surface of a semiconductor wafer. These processes achieved partial success in removing the pollutant. However, the brushes scratch the surface of the substrate, and also it may generate undesirable static electricity. And, the high-pressure liquid is apt to cut the soft surface of the substrate. Further, the high-pressure liquid has a drawback that it is not easy to withdraw the liquid from the brushes and high-pressure liquid cleaning system. Meanwhile, it is well known that solid and gas phase carbon dioxide (CO 2 snow) can remove the polluted fine particles from the surface of the substrate without the above-mentioned drawback. One of the techniques is disclosed in U.S. Pat. No. 5,125,979. In the above mentioned technique, there are provided a small expansion chamber and a large expansion chamber which are communicated with each other through a venturi interposed therebetween. At an outlet of the large expansion chamber is provided an accelerating chamber for accelerating an injecting speed of a cleaning medium. The cleaning medium is supplied from a cleaning medium supplying source to the small expansion chamber, and then adiabatically expanded while being supplied through the venturi to the large expansion chamber, thereby forming Co 2 snow having snow particles of about 46%. The Co 2 snow is accelerated by inert gas introduced to the accelerating chamber, and then injected through a nozzle to a desired position in which the cleaning process is performed. That is, in the technique, the cleaning medium is transformed into the Co 2 snow, while passing through the venturi. Then, the particles of the Co 2 snow grow, while the Co 2 snow passes through the large expansion chamber. The cleaning medium injected through the nozzle cleans the pollutant on the surface of the substrate and then sublimed. However, in the technique, since the cleaning medium of Co 2 is transformed into the Co 2 snow, while passing through one venturi, a solidification rate of the cleaning medium is low. Furthermore, since the cleaning process is typically performed at a high presser, there is a problem that a large quantity of cleaning medium is needed to remove the polluted fine particles under the same conditions. To solve the problem, the applicant had proposed Korean Patent application No. 2000-8560 filed on Feb. 22, 2000, entitled “Nozzle for cleaning components of semiconductor fabricating equipment”. As shown in FIGS. 1 to 3 , the nozzle for cleaning components of a semiconductor fabricating equipment has first and second venturi blocks 51 and 53 , which are disposed in series, to provide a wider cleaning surface than a single nozzle, thereby maximizing the cleaning efficiency. A carrier gas supplying pipe 61 is connected to a base block 55 in which the first venturi block 51 is disposed. A cleaning medium supplying pipe 59 is connected to a sub-block 57 disposed at an upper side of the base block 55 . The cleaning medium supplying pipe 59 is connected to a cleaning medium chamber 13 b in which high-pressure Co 2 is stored in liquid phase. The cleaning medium supplying pipe 59 is controlled by a regulator 11 to have a lower pressure of 100˜120 psi. Since the cleaning medium of Co 2 is reduced from the high pressure to the low pressure, the particles of snow state are formed in the cleaning medium. The cleaning medium controlled to have the above-mentioned pressure is supplied through the cleaning medium supplying pipe 59 and the sub-block 57 to a fan-shaped space 51 a formed in the base block 55 . The carrier gas supplying pipe 61 is connected to a carrier gas chamber 13 a to supply carrier gas such as N 2 to the base block 55 . The carrier gas is stored in the carrier gas chamber 13 a at a high pressure. As shown in FIG. 2 , at an distal end of the carrier gas supplying pipe 61 , there is formed a slot 61 a for uniformly injecting the carrier gas into a plurality of venturi paths formed at the first venturi block 51 . At the sub-block 57 , there is formed a path 57 a perpendicular to the fan-shaped space 51 a to be communicated with the space 51 a . The cleaning medium supplying pipe 59 has a circular portion on which the cleaning medium is dashed, so that the cleaning medium is uniformly injected into the fan-shaped space 51 a. Accordingly, the cleaning medium supplied through the cleaning medium supplying pipe 59 to the space 51 a of the base block 55 is mixed with the carrier gas injected from the carrier gas supplying pipe 61 in the space 51 a so as to firstly induce the solidification of the cleaning medium. The mixed gas is adiabatically expanded, while passing through the venturi paths formed in the first venturi block 51 , whereby a temperature and a pressure of the mixed gas are sharply reduced. Since the cleaning medium is adiabatically expanded in the first venturi block 51 , the solidification of the cleaning medium is further promoted. Further, the cleaning medium is adiabatically expanded again, while passing through the second venturi block 53 , and thus, the solidification of the cleaning medium is promoted once more. However, the conventional technique as described above has a structure that the cleaning medium supplying path is perpendicular to the carrier gas supplying path at a place where the cleaning medium and the carrier gas is mixed. Therefore, since the carrier gas having the higher pressure than the cleaning medium is flowed back to the cleaning medium supplying path, there is a problem that the cleaning medium supplying path is clogged. Furthermore, since the carrier gas is supplied at the high pressure, there is another problem that the consumption of the carrier gas is increased. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a perspective view a conventional nozzle for cleaning a component of a semiconductor; FIG. 2 is an exploded perspective view of the nozzle of FIG. 1 ; FIG. 3 is a cross-sectional view taken along the line III-III of FIG. 1 ; FIG. 4 is a perspective view of a nozzle for injecting sublimable solid particles entrained in gas for cleaning a surface according to the present invention; FIG. 5 is an exploded perspective view of the nozzle of FIG. 4 ; FIG. 6 is a plan view of the nozzle in which a sub-block is removed according to the present invention; and FIG. 7 is a cross-sectional view taken along the line VII-VII of FIG. 4 . detailed-description description="Detailed Description" end="lead"? |
Creep resistant magnesium alloy |
A magnesium based alloy consists of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, and 0-0.1% oxidation inhibiting element(s) the remainder being magnesium except for incidental impurities. |
1. A magnesium based alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, and 0-0.1% oxidation inhibiting element(s) the remainder being magnesium except for incidental impurities. 2. A magnesium alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, 0-0.1% oxidation inhibiting element(s), no more than 0.15% titanium, no more than 0.15% hafnium, no more than 0.1% aluminium, no more than 0.1% copper, no more than 0.1% nickel, no more than 0.1% silicon, no more than 0.1% silver, no more than 0.1% yttrium, no more than 0.1% thorium, no more than 0.01% iron, no more than 0.005% strontium, the balance being magnesium except for incidental impurities. 3. An alloy as claimed in claim 1 wherein the magnesium content is 95.5-97% by weight. 4. An alloy as claimed in claim 1 wherein the neodymium content is 1.6-1.8% by weight. 5. An alloy as claimed in claim 1 wherein the content of rare earth(s) other than neodymium is 0.9-1.1% by weight. 6. An alloy as claimed in claim 1 which contains a plurality of rare earth elements other than neodymium and in which cerium comprises over half the weight of the rare earth elements other than neodymium. 7. An alloy as claimed in claim 1 wherein the zirconium content is greater than 0.4% by weight. 8. An alloy as claimed in claim 1 wherein the zinc content is 0.4-0.6% by weight. 9. A magnesium based alloy having a microstructure comprising equiaxed grains of magnesium based solid solution separated at the grain boundaries by a generally contiguous intergranular phase, the grains containing a uniform distribution of nano-scale precipitate platelets on more than one habit plane containing magnesium and neodymium, the intergranular phase consisting almost completely of rare earth elements, magnesium and a small amount of zinc, and the rare earth elements being substantially cerium and/or lanthanum. 10. A method of producing a magnesium alloy article, the method comprising subjecting to a T6 heat treatment an article cast from an alloy as claimed in claim 1. 11. A method of manufacturing a magnesium alloy article, the method comprising the steps of: (a) solidifying in a mould a casting of an alloy as claimed in claim 1, (b) heating the solidified casting at a temperature of 500-550° C. for a first period of time, (c) quenching the casting, and (d) ageing the casting at a temperature of 200-230° C. for a second period of time. 12. A method of manufacturing a casting made from magnesium alloy comprising the steps of: (i) melting an alloy as claimed in claim 1 to form a molten alloy, (ii) introducing the molten alloy into a sand mould or permanent mould and allowing the molten alloy to solidify, (iii) removing the resultant solidified casting from the mould, and (iv) maintaining the casting within a first temperature range for a first period of time during which a portion of an intergranular phase of the casting is dissolved, and subsequently maintaining the casting within a second temperature range lower than the first temperature range for a second period of time during which nano-scale precipitate platelets are caused to precipitate within grains of the casting and at grain boundaries. 13. A method as claimed in claim 12 wherein the first temperature range is 500-550° C., the second temperature range is 200-230° C., the first period of time is 6-24 hours, and the second period of time is 3-24 hours. 14. An engine block for an internal combustion engine produced by a method as claimed in claim 10. 15. An engine block for an internal combustion engine formed from a magnesium alloy as claimed in claim 1. |
<SOH> BACKGROUND TO THE INVENTION <EOH>Magnesium alloys have been used for many years in applications where the material of construction is required to exhibit a high strength to weight ratio. Typically a component made from a magnesium alloy could be expected to have a weight about 70% of an aluminium (Al) alloy component of similar volume. The aerospace industry has accordingly been a significant user of magnesium alloys and magnesium alloys are used for many components in modern defence aircraft and spacecraft. However, one limitation preventing wider use of magnesium alloys is that, when compared to aluminium alloys, they typically have poorer resistance to creep at elevated temperatures. With the increasing needs to control international fuel consumption and reduce harmful emissions into the atmosphere, automobile manufacturers are being pressured into developing more fuel efficient vehicles. Reducing the overall weight of the vehicles is a key to achieving this goal. A major contributor to the weight of any vehicle is the engine itself, and the most significant component of the engine is the block, which makes up 20-25% of the total engine weight. In the past significant weight savings were made by introducing an aluminium alloy block to replace the traditional grey iron block, and further reductions of the order of 40% could be achieved if a magnesium alloy that could withstand the temperatures and stresses generated during engine operation was used. However, the development of such an alloy, which combines the desired elevated temperature mechanical properties with a cost effective production process, is necessary before a viable magnesium engine block manufacturing line could be considered. In recent years, the search for an elevated temperature magnesium alloy has focused primarily on the high pressure die casting (HPDC) processing route and several alloys have been developed. HPDC was considered to be the best option for achieving the high productivity rates required to counteract the probable high cost of the base magnesium alloy. However, HPDC is not necessarily the best process for the manufacture of an engine block and, in reality, the majority of blocks are still precision cast by gravity or low pressure sand casting. There are two major classes of magnesium sand casting alloys. (A) Alloys based on the magnesium-aluminium binary system, often with small additions of zinc (Zn) for improved strength and castability. These alloys have adequate room temperature mechanical properties, but do not perform well at elevated temperatures and are inappropriate at temperatures in excess of 150° C. These alloys do not contain expensive alloying elements and are widely used in areas where high temperature strength is not a requirement. (B) Alloys able to be grain refined by the addition of zirconium (Zr). The major alloying elements in this group are zinc, yttrium (Y), silver (Ag), thorium (Th), and the rare earth (RE) elements such as neodymium (Nd). Throughout this specification the expression “rare earth” is to be understood to mean any element or combination of elements with atomic numbers 57 to 71, ie. lanthanum (La) to lutetium (Lu). With the right choice of alloying additions, alloys in this group can have excellent room and elevated temperature mechanical properties. However, with the exception of zinc, the alloying additions within this group, including the grain refiner, are expensive with the result that the alloys are generally restricted to aeronautical applications. The magnesium alloy ML10, developed in the USSR, has been used for many years for cast parts intended for use in aircraft at temperatures up to 250° C. ML10 is a high strength magnesium alloy developed on the basis of the Mg—Nd—Zn—Zr system. ML19 alloy additionally contains yttrium. A paper by Mukhina et al entitled “Investigation of the Microstructure and Properties of Castable Neodymium and Yttrium-Bearing Magnesium Alloys at Elevated Temperatures” published in “Science and Heat Treatment” Vol 39, 1997, indicated typical compositions (% by weight) of ML10 and ML19 alloys are: ML10 ML19 Nd 2.2-2.8 1.6-2.3 Y Nil 1.4-2.2 Zr 0.4-1.0 0.4-1.0 Zn 0.1-0.7 0.1-0.6 Mg Balance Balance with impurity levels of: Fe <0.01 Si <0.03 Cu <0.03 Ni <0.005 Al <0.02 Be <0.01 Alternatives which have been developed are alloys known to those in the art as QE22 (an Mg—Ag—Nd—Zr system alloy) and EH21 (an Mg—Nd—Zr—Th system alloy). However, these alternatives are expensive to manufacture as they contain significant quantities of silver and thorium respectively. Heat resistant grain refined magnesium alloys can be strengthened by a T6 heat treatment which comprises an elevated temperature solution treatment, followed by quenching, followed by an artificial aging at an elevated temperature. In heating before quenching the excess phases pass into solid solution. In the aging process refractory phases, in the form of finely dispersed submicroscopic particles, are segregated and these create microheterogeneities inside the grains of the solid solution, blocking diffusion and shear processes at elevated temperatures. This improves the mechanical properties, namely the ultimate long term strength and the creep resistance of the alloys at high temperature. To date, a sand casting magnesium alloy having desired elevated temperature (eg 150-200° C.) properties at a reasonable cost has been unavailable. At least preferred embodiments of the present invention relate to such an alloy and the present invention is particularly, but not exclusively, directed to application with precision casting operations. |
<SOH> SUMMARY OF THE INVENTION <EOH>In a first aspect the invention provides a magnesium based alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, and 0-0.1% oxidation inhibiting element(s), the remainder being magnesium except for incidental impurities. In a second aspect, the present invention provides a magnesium alloy consisting of, by weight: 1.4-1.9% neodymium, 0.8-1.2% rare earth element(s) other than neodymium, 0.4-0.7% zinc, 0.3-1% zirconium, 0-0.3% manganese, 0-0.1% oxidation inhibiting element, no more than 0.15% titanium, no more than 0.15% hafnium, no more than 0.1% aluminium, no more than 0.1% copper, no more than 0.1% nickel, no more than 0.1% silicon, no more than 0.1% silver, no more than 0.1% yttrium, no more than 0.1% thorium, no more than 0.01% iron, no more than 0.005% strontium, the balance being magnesium except for incidental impurities. Preferably, alloys according to the second aspect of the present invention: (a) contain less than 0.1% titanium, more preferably less than 0.05% titanium, more preferably less than 0.01% titanium, and most preferably substantially no titanium; (b) contain less than 0.1% hafnium, more preferably less than 0.05% hafnium, more preferably less than 0.01% hafnium, and most preferably substantially no hafnium; (c) contain less than 0.05% aluminium, more preferably less than 0.02% aluminium, more preferably less than 0.01% aluminium, and most preferably substantially no aluminium; (d) contain less than 0.05% copper, more preferably less than 0.02% copper, more preferably less than 0.01% copper, and most preferably substantially no copper; (e) contain less than 0.05% nickel, more preferably less than 0.02% nickel, more preferably less than 0.01% nickel, and most preferably substantially no nickel; (f) contain less than 0.05% silicon, more preferably less than 0.02% silicon, more preferably less than 0.01% silicon, and most preferably substantially no silicon; (g) contain less than 0.05% silver, more preferably less than 0.02% silver, more preferably less than 0.01% silver, and most preferably substantially no silver; (h) contain less than 0.05% yttrium, more preferably less than 0.02% yttrium, more preferably less than 0.01% yttrium, and most preferably substantially no yttrium; (i) contain less than 0.05% thorium, more preferably less than 0.02% thorium, more preferably less than 0.01% thorium, and most preferably substantially no thorium; (j) contain less than 0.005% iron, most preferably substantially no iron; and (k) contain less than 0.001% strontium, most preferably substantially no strontium. Preferably, alloys according to the present invention contain at least 95% magnesium, more preferably 95.5-97% magnesium, and most preferably about 96.3% magnesium. Preferably, the neodymium content is greater than 1.5%, more preferably greater than 1.6%, more preferably 1.6-1.8% and most preferably about 1.7%. The neodymium content may be derived from pure neodymium, neodymium contained within a mixture of rare earths such as a misch metal, or a combination thereof. Preferably, the content of rare earth(s) other than neodymium is 0.9-1.1%, more preferably about 1%. Preferably, the rare earth(s) other than neodymium are cerium (Ce), lanthanum (La), or a mixture thereof. Preferably, cerium comprises over half the weight of the rare earth elements other than neodymium, more preferably 60-80%, especially about 70% with lanthanum comprising substantially the balance. The rare earth(s) other than neodymium may be derived from pure rare earths, a mixture of rare earths such as a misch metal or a combination thereof. Preferably, the rare earths other than neodymium are derived from a cerium misch metal containing cerium, lanthanum, optionally neodymium, a modest amount of praseodymium (Pr) and trace amounts of other rare earths. The habit plane of the precipitating phase in Mg—Nd—Zn alloys is related to the zinc content, being prismatic at very low levels of Zn and basal at levels in excess of about 1 wt %. The best strength results are obtained at zinc levels which promote a combination of the two habit planes. Preferably, the zinc content is less than 0.65%, more preferably 0.4-0.6%, more preferably 0.45-0.55%, most preferably about 0.5%. Reduction in iron content can be achieved by addition of zirconium which precipitates iron from molten alloy. Accordingly, the zirconium contents specified herein are residual zirconium contents. However, it is to be noted that zirconium may be incorporated at two different stages. Firstly, on manufacture of the alloy and secondly, following melting of the alloy just prior to casting. The elevated temperature properties of alloys of the present invention are reliant on adequate grain refinement and it is therefore necessary to maintain a level of zirconium in the melt beyond that required for iron removal. For desired tensile and compressive strength properties the grain size is preferably less than 200 μm and more preferably less than 150 μm. The relationship between creep resistance and grain size in alloys of the present invention is counter-intuitive. Conventional creep theory will predict that the creep resistance will decrease as the grain size decreases. However, alloys of the present invention have shown a minimum in creep resistance at a grain size of 200 μm and improvements in creep resistance at smaller grain sizes. For optimum creep resistance the grain size is preferably less than 100 μm and more preferably about 50 μm. Preferably, the zirconium content will be the minimum amount required to achieve satisfactory iron removal and adequate grain refinement for the intended purpose. Typically, the zirconium content will be greater than 0.4%, preferably 0.4-0.6%, more preferably about 0.5%. Manganese is an optional component of the alloy which may be included if there is a need for additional iron removal over and above that achieved by zirconium, especially if the zirconium levels are relatively low, for example below 0.5 wt %. Elements which prevent or at least inhibit oxidation of molten alloy, such as beryllium (Be) and calcium (Ca), are optional components which may be included especially in circumstances where adequate melt protection through cover gas atmosphere control is not possible. This is particularly the case when the casting process does not involve a closed system. Ideally, the incidental impurity content is zero but it is to be appreciated that this is essentially impossible. Accordingly, it is preferred that the incidental impurity content is less than 0.15%, more preferably less than 0.1%, more preferably less than 0.01%, and still more preferably less than 0.001%. In a third aspect, the present invention provides a magnesium based alloy having a microstructure comprising equiaxed grains of magnesium based solid solution separated at the grain boundaries by a generally contiguous intergranular phase, the grains containing a uniform distribution of nano-scale precipitate platelets on more than one habit plane containing magnesium and neodymium, the intergranular phase consisting almost completely of rare earth elements, magnesium and a small amount of zinc, and the rare earth elements being substantially cerium and/or lanthanum. The grains may contain clusters of small spherical and globular precipitates. The spherical clusters may comprise fine rod-like precipitates. The globular precipitates may be predominantly zirconium plus zinc with a Zr:Zn atomic ratio of approximately 2:1. The rod-like precipitates may be predominantly zirconium plus zinc with a Zr:Zn atomic ratio of approximately 2:1. The expression “generally contiguous” as used in this specification is intended to mean that at least most of the intergranular phase is contiguous but that some gaps may exist between otherwise contiguous portions. In a fourth aspect, the present invention provides a method of producing a magnesium alloy article, the method comprising subjecting to a T6 heat treatment an article cast from an alloy according to the first, second or third aspect of the present invention. In a fifth aspect, the present invention provides a method of manufacturing a magnesium alloy article, the method comprising the steps of: (a) solidifying in a mould a casting of an alloy according to the first, second or third aspects of the present invention, (b) heating the solidified casting at a temperature of 500-550° C. for a first period of time, (c) quenching the casting, and (d) ageing the casting at a temperature of 200-230° C. for a second period of time. Preferably, the first period of time is 6-24 hours and the second period of time is 3-24 hours. In a sixth aspect, the present invention provides a method of manufacturing a casting made from magnesium alloy comprising the steps of: (i) melting an alloy according to the first, second or third aspects of the present invention to form a molten alloy, (ii) introducing the molten alloy into a sand mould or permanent mould and allowing the molten alloy to solidify, (iii) removing the resultant solidified casting from the mould, and (iv) maintaining the casting within a first temperature range for a first period of time during which a portion of an intergranular phase of the casting is dissolved, and subsequently maintaining the casting within a second temperature range lower than the first temperature range for a second period of time during which nano-scale precipitate platelets are caused to precipitate within grains of the casting and at grain boundaries. The first temperature range is preferably 500-550° C., the second temperature range is preferably 200-230° C., the first period of time is preferably 6-24 hours, and the second period of time is preferably 3-24 hours. In a seventh aspect, the present invention provides an engine block for an internal combustion engine produced by a method according to the fourth, fifth or sixth aspect of the present invention. In an eighth aspect, the present invention provides an engine block for an internal combustion engine formed from a magnesium alloy according to the first, second or third aspects of the present invention. Specific reference is made above to engine blocks but it is to be noted that alloys of the present invention may find use in other elevated temperature applications as well as low temperature applications. detailed-description description="Detailed Description" end="lead"? |
Alzheimer's disease diagnosis based on mitogen-activated protein kinase phosphorylation |
A method of diagnosing Alzheimer's disease in a patient comprises determining whether the phosphorylation level of an indicator protein in cells of the patient after stimulus with an activator compound is abnormally elevated as compared to a basal phosphorylation level, the indicator protein being e.g. Erk1/2 and the activator compound being e.g. bradykinin. |
1. A method of diagnosing Alzheimer's disease in a subject, said method comprising: (a) measuring a basal level of phosphorylation of an indicator protein in cells from the subject; (b) contacting cells from the subject with an activator compound, the activator compound and indicator protein being selected such that the activator elicits a differential response of activated phosphorylation of the indicator protein in cells of the subject as compared to an activated phosphorylation response in cells from a non-Alzheimer's control subject at a predetermined time after the contacting is initiated; (c) measuring an activated phosphorylation level of the indicator protein in said subject cells at the predetermined time after contacting is initiated; and (d) calculating a ratio of the activated phosphorylation level determined in step (c) to the basal phosphorylation level of step (a); and (e) comparing the calculated ratio of step (d) to previously determined activated phosphorylation ratios measured from known Alzheimer's disease cells and from known non-Alzheimer's disease cells at said predetermined time; wherein if the calculated ratio is not statistically different from the previously determined ratios for said known Alzheimer's disease cells, the diagnosis is positive, and/or if the calculated ratio is not statistically different from the previously determined ratios for said known non-Alzheimer's disease cells, the diagnosis is negative. 2. A method of diagnosing Alzheimer's disease in a subject comprising: (a) in cells of the subject, measuring a background phosphorylation level of an indicator calcium signaling pathway protein whose phosphorylation is associated with IP-3R-sensitive Ca2+ elevation in the cells; (b) stimulating cells of the subject by contact with and IP3R agonist that elicits a differential response in the phosphorylation level of the indicator protein in cells of Alzheimer's subjects as compared to the level in a non-Alzheimer's control cell; (c) thereafter, measuring a response phosphorylation level of the indicator protein in the contacted cells, and (d) determining whether the response phosphorylation level of the indicator protein as compared to the background level matches the response level known for a cell from an Alzheimer's subjects or from a healthy control cell. 3. The method of claim 2, comprising first measuring the background phosphorylation level of the indicator protein in a culture of cells, then adding the IP3R agonist to the culture, and measuring the response phosphorylation level. 4. The method of claim 2, comprising measuring the background level in a first aliquot of cells, stimulating a similar aliquot of the cells, and measuring the response level in the aliquot. 5. The method of claim 2, wherein the measuring comprises an immunoassay 6. The method of claim 2, wherein the measuring comprises an immunoassay of disrupted cells. 7. The method of claim 2, wherein the IP3-R agonist is selected from the group consisting of bradykinin, bombesin, choleystokinin, thrombin, prostaglandin F2α and vasopressin. 8. The method of claim 2, wherein said cells are selected from the group consisting of fibroblasts, buccal mucosal cells, neurons, and blood cells. 9. A method of diagnosing Alzheimer's disease in a subject, comprising: (a) obtaining cells from said subject; (b) measuring the basal level of phosphorylation of an indicator protein in said cells; (c) contacting said cells with an activator of phosphorylation of the indicator protein; (d) measuring the phosphorylation level of the indicator protein in said cells at a predetermined time after initiation of the contacting; and (e) calculating a first ratio of the level measured in step (d) to the level measured in step (b) and comparing said first ratio to a previously determined second ratio of said levels obtained at said predetermined time from known Alzheimer's disease cells and a third ratio of said levels obtained at said predetermined time from known non-Alzheimer's disease cells; wherein (i) if the first ratio of step (e) is statistically not different from the previously determined second ratio, the diagnosis is positive, and (ii) if the first ratio of step (e) is not statistically different from the previously determined third ratio, the diagnosis is negative. 10. The method of claim 9, wherein the predetermined time of step (d) is a time when the difference between the second ratio and the third ratio of step (e) is greatest. 11. The method of claim 1, wherein the activator is selected from the group consisting of bradykinin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin. 12. The method of claim 1 wherein the measuring is by immunoassay, and wherein said subject's cells are contacted with an antibody specific for the phosphorylated indicator protein, permitting the antibody to bind to the indicator protein, and detecting the antibody bound to the indicator protein. 13. The method of claim 12 wherein said immunoassay is a radioimmunoassay a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay. 14-17 (canceled). 18. A method of diagnosing Alzheimer's disease in a subject, comprising: contacting cells from the subject with an agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP3) receptor, measuring the amount of phosphorylation of a MAPK protein in the subject's cells at one or more time points after the contacting step, and comparing the amount of phospliorylation of the MAPK protein in the subject's cells at the one or more time points with the amount of phosphorylation in cells from a non-Alzheimer's control subject at the same time points after contacting the control cells with the agent, wherein increased phosphorylation of the MAPK protein in the subject's cells compared to the control cells is diagnostic of Alzheimer's disease. 19. The method of claim 18, wherein the agent is bradykinin or a bradykinin receptor agonist. 20. The method of claim 18, wherein the agent is bombesin. 21. The method of claim 18, wherein the agonist is one which induces IP3-mediated Ca2+ release. 22. The method of claim 18, wherein the MAPK protein is Erk1/2. 23. The method of claim 18, wherein the amount of phosphorylation is measured at a single time point after the contacting step. 24. The method of claim 18, wherein the measuring comprises measuring the amount of phosphorylation in a first aliquot of the subject's cells at a first time point after the contacting step, and measuring the amount of phosphorylation in a second aliquot of the subject's cells at a second time point after the contacting step. 25. The method of claim 18, wherein the time points are selected from one or more of about 0.5 minutes, 1 minute, 2 minutes, 2.5 minutes, 5 minutes, 10 minutes, 20, and 30 minutes. 26. he method of claim 18, wherein the cells are from peripheral tissue. 27. The method of claim 18, wherein the cells are skin fibroblasts. 28. The method of claim 18, wherein the measuring step comprises detecting phosphorylation in a lysate of the subject's cells. 29. The method of claim 18, wherein the measuring step is carried out in vitro. 30. The method of claim 18, wherein the measuring step comprises gel electrophoresis. 31. The method of claim 18, wherein the measuring step comprises Western blotting. 32. The method of claim 31, wherein the Western blotting comprises using an anti-phospho-MAP kinase antibody. 33. The method of claim 18, wherein the increased phosphorylation is an elevation in the amount of phosphorylated protein at a single time point. 34. The method of claim 18, wherein the increased phosphorylation is an increase in duration of the phosphorylated protein. 35. The method of claim 18, wherein the subject lacks clinical manifestations of Alzheimer's disease. 36. The method of claim 18, further comprising contacting the subject's cells with one or more inhibitors selected from the group consisting of an inhibitor of protein kinase C activity, an inhibitor of PI-3 kinase activity, an inhibitor of C-SRC protein tyrosine kinase activity, an inhibitor of the IP-3 receptor and an inhibitor of a protein phosphatase. 37. The method of claim 18, wherein the increased phosphorylation is inhibited by contacting the subject's cells with an inhibitor selected from the group consisting of an inhibitor of protein kinase C activity, C-src protein tyrosine kinase activity, PI-3 kinase activity, and the IP-3 receptor. 38. The method of claim 37, wherein said inhibitor is selected from the group consisting of BiSM-1, PP1, and 2-aminoethoxydiphenyl borate. 39-57. (Canceled). |
<SOH> BACKGROUND OF THE INVENTION <EOH>The invention provides a diagnostic and screening test for Alzheimer's disease (“AD”). An example of the test involves detecting abnormally enhanced phosphorylation of extracellular signal-regulated kinase type 1 or 2 (“Erk1/2”) in skin fibroblasts from AD patients after stimulating the cells with agonist such as bradykinin or other agents that stimulate the inositol 1,4,5-trisphosphate (IP3) receptor, in comparison to cells from age-matched controls. Enhanced phosphorylation may be measured by Western blot using antibodies specific for the phosphorylated protein or other similar approaches. Accumulating evidence indicates that the early pathogenesis of Alzheimer's disease (AD) involves perturbation of intracellular calcium homeostasis and increased levels of oxidative stress that contribute to excitatory toxicity and neuronal death in the AD brain (Putney, 2000; Yoo et al., 2000; Sheehan et al., 1997). Studies have reported enhanced elevation of intracellular Ca 2+ levels in AD brains as well as in peripheral cells in response to activation of bradykinin receptors and inactivation of a K + channel (Ito et al., 1994; Etcheberrigaray et al., 1994; Hirashima, et al., 1996; Gibson et al., 1996; Etcheberrigaray et al., 1998). Critical proteins such as amyloid precursor protein (APP), presenilin 1 and presenilin 2, mutations of which are associated with the pathogenesis of AD, have been reported to induce dysregulation of both the IP3 receptor (IP3R) and the ryanodine receptor-(RYR-) mediated intracellular Ca 2+ homeostasis (Yoo et al., 2000; Leissring et al., 1999; 2000; Mattson et al., 2000; Barrow et al., 2000). The alteration in cytosolic Ca 2+ concentration is thought to contribute to the pathophysioloy of AD, including increased production of the neurotoxic 42 amino acid β-amyloid peptide (APβ) involved in plaque formation, hyperphosphorylation of tau protein involved in formation of neurfibrillay tangles, and enhanced general vulnerability of neurons to cell death. Bradykinin (BK) is a potent vasoactive nonapeptide that is generated in the course of various inflammatory conditions. BK binds to and activates specific cell membrane BK receptor(s), thereby triggering a cascade of intracellular events leading to the phosphorylation of proteins known as “mitogen activated protein kinase” (MAPK; see below). Phosphorylation of proteins, the addition of a phosphate group to a Ser, Thr or Tyr residue, is mediated by a large number of enzymes known collectively as protein kinases. Phosphorylation normally modifies the function of, and usually activates, a protein. Homeostasis requires that phosphorylation be a transient process, which is reversed by phosphatase enzymes that dephosphorylate the substrate. Any aberration in phosphorylation or dephosphorylation disrupts biochemical pathways and multiple cellular functions. Such disruptions may be the basis for certain brain diseases. Increased intracellular Ca 2+ levels in response to BK is mediated at least by the “type 2” BK receptor (BKb2R), a G-protein-coupled receptor. Stimulation of BKb2R by BK activates phospholipase C (PLC) resulting in production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), second messengers involved in regulation of intracellular Ca 2+ levels and activation of protein kinase C (PKC). The PLC/phospholipid/PKC pathway also interact with the Ras signaling pathway that activates the MAPK pathway. MAPK (or MAP kinase) refers to an enzyme family termed “mitogen activated protein kinase,” an important member of which is the “extracellular signal-regulated kinase” type 1 or 2 (“Erk1/2”) (Berridge, 1984; Bassa et al., 1999). Erk1/2 receive signals from multiple signal transductional pathways and is part of a pathway that leads to cell proliferation and differentiation by regulation of gene expression through a number of transcriptional factors including cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Erk1/2 phosphorylates tau protein at multiple Ser/Thr sites including Ser262 and Ser356 (Reynolds et al., 2000), which are in microtubule-binding regions of tau. Phosphorylation of Ser262 markedly compromises the ability of tau to assemble and stabilize microtubules (Biemat et al., 1993; Lu et al., 1993). Increased oxidative stress, aberrant expression of amyloid precursor protein (APP), and exposure to APβ cause activation of MAPK (McDonald et al., 1998; Ekinci and Shea, 1999; Grant et al., 1999) and enhanced tau phosphorylation (Greenberg et al., 1994). Young L T et al., Neurosci Lett, 1988, 94:198-202 studied IP3 receptor binding sites in autopsied brains from 10 subjects with AD and 10 age-matched controls. In the parietal cortex and hippocampus, there was a 50-70% loss of [ 3 H]-IP3 binding whereas no significant changes were observed in frontal, occipital and temporal cortices, caudate or amygdala. Scatchard analysis confirmed a reduction in receptor density rather than a change in affinity. Also, many neurotransmitters, hormones and growth factors act at membrane receptors to stimulate the phosphodiesterase hydrolysis of phosphatidyl-inositol 4,5-bisphosphate (PIP2) generating the comessengers IP3 and diacylglycerol (DAG). DAG stimulates PKC while IP3 was initially postulated to activate specific receptors leading to release of intracellular calcium, probably from the endoplasmic reticulum. Though earlier reports had detected 32 P-IP3 binding to liver and adrenal microsomes and to permeabilized neutrophils and liver cells, Solomon Snyder's group was the first to localize, isolate, analyze and later clone, IP3 receptors. Worley P F et al., Nature 1987;325:159-161, demonstrated high affinity, selective binding sites for 3 H- and 32 P-labelled IP3 in the brain at levels 100-300 times higher than those observed in peripheral tissues. These receptors were considered physiologically relevant because the potencies of various myoinositol analogues at the IP3 binding site corresponded to their potencies in releasing calcium from microsomes. Brain autoradiograms demonstrated discrete, heterogeneous localization of IP3 receptors. In 1988, this group (Supattapone S et al., J Biol Chem, 1988, 263:1530-1534, reported the solubilization, purification to homogeneity, and characterization of an IP3 receptor from rat cerebellum. The purified receptor was a globular protein that migrated in electrophoresis as one protein band with an Mr of 260 kDa. In a review, Snyder et al. ( Cell Calcium, 1989, 10:337-342) noted that immunohistochemical studies with antisera to the purified receptor protein localized the receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane. The IP3 receptor protein was selectively phosphorylated by cAMP-dependent protein kinase. This phosphorylation decreased 10-fold the potency of IP3 in releasing calcium from brain membranes. Ferris C D et al., Proc Natl Acad Sci USA, 1991 88:2232-2235 later studied phosphorylation of IP3 receptors with purified receptor protein reconstituted in liposomes (to remove detergent that can inhibit protein kinases). The IP3 receptor was stoichiometrically phosphorylated by protein kinase C (PKC) and CaM kinase II as well as by protein kinase A (PKA). IP3 receptors are regulated by phosphorylation catalyzed by the three enzymes which was additive and involved different peptide sequences. Phosphorylation by (1) PKC which was stimulated by Ca 2+ and DAG, and (2) by CaM kinase II which required Ca 2+ , provided a means whereby Ca 2+ and DAG, formed during inositol phospholipid turnover, regulate IP3 receptors. Chadwick C C et al., Proc Natl Acad Sci USA, 1990 87:2132-2136, described the isolation from smooth muscle of an IP3 receptor that was an oligomer of a single polypeptide with a Mr of 224 kDa. Furuichi T, et al., FEBS Lett, 1990 267:85-88 examined distribution of IP3 receptor mRNA in mouse tissues. The concentration of was greatest in cerebellar tissue. Moderate amounts of IP3 receptor mRNA were present in other brain tissue: thymus, heart, lung, liver, spleen, kidney, and uterus. Small amounts of IP3 receptor mRNA were observed in skeletal muscle and testicular tissue. Based on in situ hybridization, a considerable amount of IP3 receptor mRNA was located in smooth muscle cells, such as those of the arteries, bronchioles, oviduct and uterus. Ferris C D et al., J Biol Chem, 1992, 267:7036-7041, demonstrated serine autophosphorylation of the purified and reconstituted IP3 receptor and found serine protein kinase activity of the IP3 receptor toward a specific peptide substrate. The investigators concluded that the IP3 receptor protein and the phosphorylating activity reside in the same molecule. Ross C A et al. ( Proc Natl Acad Sci USA, 1992, 89:4265-4269), cloned three IP3R cDNAs, designated IP3R-II, -III, and -IV, from a mouse placenta cDNA library. All three displayed strong homology in membrane-spanning domains M7 and M8 to the originally cloned cerebellar IP3R-I, with divergences predominantly in cytoplasmic domains. Levels of mRNA for the three additional IP3Rs in general were substantially lower than for IP3R-I, except for the gastrointestinal tract where levels were comparable. Cerebellar Purkinje cells expressed at least two and possibly three distinct IP3Rs, suggesting heterogeneity of IP3 action within a single cell. Sharp A H, Neuroscience, 1993, 53:927-42, examined in detail the distribution of IP3 receptors in the rat brain and spinal cord using immunohistochemical methods. IP3 receptors are present in neuronal cells, fibers and terminals in a wide distribution of areas throughout the CNS, including the olfactory bulb, thalamic nuclei and dorsal hom of the spinal cord, in circumventricular organs and neuroendocrine structures such as the area postrema, choroid plexus, subcommisural organ, pineal gland and pituitary. Ca2+ release mediated by the phosphoinositide second messenger system is important in control of diverse physiological processes. Studies of IP3 receptors in lymphocytes (T cells) by Snyder's group localized these receptors to the plasma membrane. Capping of the T cell receptor-CD3 complex, which is associated with signal transduction, was accompanied by capping of IP3 receptors. The IP3 receptor on T cells appears to be responsible for the entry of Ca 2+ that initiates proliferative responses (Khan, A A et al., Science, 1992, 257:815-818) Further with regard to IP3, Wilcox R A et al., Trends Pharmacol Sci, 1998, 19:467-475, noted that receptor-mediated activation of PLC to generate IP3 is a ubiquitous signalling pathway in mammalian systems. A family of three IP3 receptor subtype monomers form functional tetramers, which act as IP3 effectors, providing a ligand-gated channel that allows Ca 2+ ions to move between cellular compartments. As IP3 receptors are located principally, although not exclusively, in the endoplasmic reticular membrane, IP3 is considered to be a second messenger that mobilizes Ca 2+ from intracellular stores contributing to a variety of physiological and pathophysiological phenomena. Patel S et al., Cell Calcium, 1999, 25:247-264, reviewed the molecular properties of IP3 receptors. Several Ca 2+ -binding sites and a Ca 2+ -calmodulin-binding domain were mapped within the type I IP3 receptor, and studies on purified cerebellar IP3 receptors suggested a second Ca 2+ -independent calmodulin-binding domain. Overexpression of IP3 receptors provided further clues to the regulation of individual IP3 receptor isoforms present within cells, and the role that they play in the generation of IP3-dependent Ca 2+ signals. IP3 receptors may be involved in cellular processes such as proliferation and apoptosis. Abdel-Latif AA. Exp Biol Med (Maywood) 2001 March;226(3):153-63 reviewed evidence, both from nonvascular and vascular smooth muscle, for cross talk between the cyclic nucleotides, cAMP and cGMP via their respective protein kinases, and the Ca 2+ -dependent- and Ca 2+ -independent-signaling pathways involved in agonist-induced contraction. These included the IP3-Ca 2+ -CaM-myosin light chain kinase (MLCK) pathway and the Ca 2+ -independent pathways, including PKC, MAP kinase, and Rho-kinase. Mikoshiba K et al., Sci STKE 2000 Sep. 26;2000(51):P, described the regulated release of calcium from intracellular stores by the IP3 receptor and the relationship of this release mechanism to calcium influx from the extracellular milieu through store-operated calcium channels. They disclosed a model of functional and physical coupling of intracellular and plasma membrane calcium channels. Although AD is well known for its severe brain damage and memory loss, pathological changes are manifest elsewhere in the body and can be detected at the cellular level. Skin fibroblasts lying in the deep layer of skin reveal characteristic cellular and molecular abnormalities of AD damage. Skin fibroblasts are readily obtained and cultured for diagnostic purposes (U.S. Pat. No. 6,107,050, “Diagnostic Test for Alzheimer's Disease,” issued Aug. 22, 2000, which is incorporated herein by reference). However, there is a need for simpler, more economical, accurate and reliable methods for diagnosis of Alzheimer's disease. It is known e.g. from U.S. Pat. No. 6,107,050, Alkon et al., that differential effects of an activator of intracellular Ca2+ release can be measured. Both healthy and Alzheimer's cell types exhibit a release of calcium from storage, but Alzheimer's cells exhibit a much greater release. Known methods for measuring the release of Ca2+(i) include fluorescent indicators, absorbance indicators or a Ca2+“patch clamp” electrode, and others, and such methods may be used for diagnostic purposes. However, there is a tremendous need for more effective techniques for measuring the differential effects of IP3R activators, for diagnostic, research, and clinical purposes. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a method of diagnosing Alzheimer's disease in a patient comprising detecting the presence or absence of an abnormally elevated level of a phosphorylated indicator protein in cells of the patient after activating the cells with a compound that stimulates phosphorylation of the indicator protein, the presence of such an elevated level indicating a positive diagnosis for Alzheimer's disease. The diagnostic method comprises measuring a phosphorylation level of an indicator protein in cells of the patient at a predetermined time after stimulating the cells with an activator compound, and determining comparing the stimulis abnormally elevated as compared to a basal phosphorylation level without stimulus. The invention provides a method of diagnosing Alzheimer's disease in a subject, said method comprising: (a) measuring a basal level of phosphorylation of an indicator protein in cells from the subject; (b) contacting cells from the subject with an activator compound, the activator compound and indicator protein being selected such that the activator elicits a differential response of activated phosphorylation of the indicator protein in cells of the subject as compared to an activated phosphorylation response in cells from a non-Alzheimer's control subject at a predetermined time after the contacting is initiated; (c) measuring an activated phosphorylation level of the indicator protein in said subject cells at the predetermined time after contacting is initiated; and (d) calculating a ratio of the activated phosphorylation level determined in step (c) to the basal phosphorylation level of step (a); and (e) comparing the calculated ratio of step (d) to previously determined activated phosphorylation ratios measured from known Alzheimer's disease cells and from known non-Alzheimer's disease cells at said predetermined time; wherein if the calculated ratio is not statistically different from the previously determined ratios for said known Alzheimer's disease cells, the diagnosis is positive, and/or if the calculated ratio is not statistically different from the previously determined ratios for said known non-Alzheimer's disease cells, the diagnosis is negative. The invention further provides a method of diagnosing Alzheimer's disease in a subject comprising: in cells of the subject, measuring a background phosphorylation level of an indicator calcium signalling pathway protein whose phosphorylation is associated with IP-3R-sensitive Ca 2+ elevation in the cells; stimulating cells of the subject by contact with an IP3R agonist that elicits a differential response in the phosphorylation level of the indicator protein in cells of Alzheimer's subjects as compared to the level in a non-Alzheimer's control cell; thereafter, measuring a response phosphorylation level of the indicator protein in the contacted cells, and determining whether the response phosphorylation level of the indicator protein as compared to the background level matches the response level known for a cell from an Alzheimer's subjects or from a healthy control cell. The methods may comprise first measuring the background phosphorylation level of the indicator protein in a culture of cells, then adding the IP3R agonist to the culture, and measuring the response phosphorylation level. Or the method may comprise measuring the background level in a first aliquot of cells, stimulating a similar aliquot of the cells, and measuring the response level in the aliquot. The activator compound may be an IP3-R agonist selected from the group consisting of bradykinin, bombesin, cholecystokinin, thrombin, prostaglandin F 2α and vasopressin. The cells may be selected from the group consisting of fibroblasts, buccal mucosal cells, neurons, and blood cells. According to the invention, a method of diagnosing Alzheimer's disease in a subject comprises (a) obtaining cells from said subject; (b) measuring the basal level of phosphorylation of an indicator protein in said cells; (c) contacting said cells with an activator of phosphorylation of the indicator protein; (d) measuring the phosphorylation level of the indicator protein in said cells at a predetermined time after initiation of the contacting; and (e) calculating a first ratio of the level measured in step (d) to the level measured in step (b) and comparing said first ratio to a previously determined second ratio of said levels obtained at said predetermined time from known Alzheimer's disease cells and a third ratio of said levels obtained at said predetermined time from known non-Alzheimer's disease cells; wherein (i) if the first ratio of step (e) is statistically not different from the previously determined second ratio, the diagnosis is positive, and (ii) if the first ratio of step (e) is not statistically different from the previously determined third ratio, the diagnosis is negative. The predetermined time of step (d) may be a time when the difference between the second ratio and the third ratio of step (e) is greatest. In the inventive methods, the measuring may comprise an immunoassay of disrupted cells, and the subject's cells may be contacted with an antibody specific for the phosphorylated indicator protein, permitting the antibody to bind to the indicator protein, and detecting the antibody bound to the indicator protein. The immunoassay may be a radioimmunoassay, a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay. A further inventive method of diagnosing Alzheimer's disease in a subject comprises: (a) incubating cells from said subject with a compound in a diluent, wherein the compound stimulates calcium signaling pathway-mediated phosphorylation of an indicator protein, thereby producing stimulated cells; (b) before, at the same time or after step (a) incubating cells of the same type from the subject with a control compound or with said diluent, thereby producing unstimulated control cells; (c) comparing a level of the phosphorylated indicator protein in the stimulated cells to a level of phosphorylated indicator protein in the unstimulated control cells, wherein an increase in the level of the phosphorylated indicator protein in stimulated cells as compared to the unstimulated cells indicates the presence of Alzheimer's disease. The comparing step (c) may include the following steps: (i) contacting a protein sample from said stimulated and/or said unstimulated cells with an antibody which recognizes the phosphorylated indicator protein; and (ii) detecting the binding of said antibody to said indicator protein. The method may further comprise contacting a protein sample from said stimulated and/or said unstimulated cells with an antibody which recognizes an unphosphorylated form of said indicator protein, and detecting the binding of said antibody and unphosphorylated indicator protein, and normalizing the level of protein. The comparing step may further include the step of obtaining a protein sample from said stimulated and said unstimulated cells. A method of diagnosing the presence of Alzheimer's disease in a subject may comprise the steps of: a) stimulating cells from said subject with an activator compound that increases phosphorylation of an indicator protein, and b) comparing the level of unphosphorylated indicator protein and phosphorylated indicator protein in stimulated cells to the level of unphosphorylated indicator protein and phosphorylated indicator protein in unstimulated cells of the same type from said subject, wherein an increase in the relative level of phosphorylated indicator protein in stimulated cells as compared to unstimulated cells indicates the presence of Alzheimer's disease. The invention provides a method of diagnosing Alzheimer's disease in a subject, comprising: contacting cells from the subject with an agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP3) receptor, measuring the amount of phosphorylation of a MAPK protein in the subject's cells at one or more time points after the contacting step, and comparing the amount of phosphorylation of the MAPK protein in the subject's cells at the one or more time points with the amount of phosphorylation in cells from a non-Alzheimer's control subject at the same time points after contacting the control cells with the agent, wherein increased phosphorylation of the MAPK protein in the subject's cells compared to the control cells is diagnostic of Alzheimer's disease. In the methods of the invention, the agent may be bradykinin or a bradykinin receptor agonist, or bombesin, and may be an agonist which induces IP3-mediated Ca 2+ release. The amount of phosphorylation may be measured at a single time point after the contacting step. According to the invention, the measuring may comprise measuring the amount of phosphorylation in a first aliquot of the subject's cells at a first time point after the contacting step, and measuring the amount of phosphorylation in a second aliquot of the subject's cells at a second time point after the contacting step. The time points may be about 0.5 minutes or shorter, 1 minute, 2 minutes, 2.5 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, or 1 hour, or longer for some combinations of cell types, activators, and indicator proteins. The cells are typically from peripheral tissue, such as skin fibroblasts. The measuring step in the inventive methods optionally comprises detecting phosphorylation in a lysate of the subject's cells, in vitro, and may comprise gel electrophoresis, Western blotting, using an anti-phospho-MAPK antibody and/or an anti-regular MAPK protein antibody. The increased phosphorylation may be an elevation in the amount of phosphorylated protein at a single time point or an increase in duration of the phosphorylated protein The methods are effective in diagnosis where the subject lacks clinical manifestations of Alzheimer's disease. According to the invention, the methods may further comprise contacting the subject's cells with one or more inhibitors selected from the group consisting of an inhibitor of protein kinase C activity, an inhibitor of PI-3 kinase activity, an inhibitor of C-src protein tyrosine kinase activity, an inhibitor of the IP-3 receptor and an inhibitor of a protein phosphatase. Also, by way of characterizing a particularly discriminating embodiment of the invention, the methods may be characterized as having the increased phosphorylation inhibited by contacting the subject's cells with an inhibitor selected from the group consisting of an inhibitor of protein kinase C activity, C-src protein tyrosine kinase activity, PI-3 kinase activity, and the IP-3 receptor. In such methods said inhibitor can be selected from the group consisting of BiSM-1, PP1, and 2-aminoethoxydiphenyl borate. The invention further provides a method for screening compounds to identify a compound useful for treatment or prevention of Alzheimer's disease comprising: contacting test cells from an AD subject with a compound being screened, before, during, or after the contacting step, stimulating the test cells with an agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP3) receptor, measuring the amount of phosphorylation of a MAPK protein in the test cells at one or more time points after stimulating the test cells, comparing the amount of phosphorylation of the MAPK protein in the test cells at the one or more time points with the amount of phosphorylation at the same one or more time points in control cells from an AD subject that are not contacted with the compound. The methods may further comprise accepting a compound that inhibits or prevents the increased phosphorylation as a lead compound, and rejecting a compound that does not inhibit or prevent the increased phosphorylation. As in all the methods of the invention, the agent may be bradykinin or a bradykinin receptor agonist and the the MAPK protein may be Erk1/2. The methods may comprise measuring the amount of phosphorylation at a single time point after the contacting step. A further embodiment provides a method of screening compounds for usefulness as activator compounds in a stimulus-response assay, comprising measuring the effect of the compound on phosphorylation of an indicator protein in AD cells and control cells and selecting a compound that increases phosphorylation of the indicator protein in amount and/or duration in AD cells as compared to control cells. Another embodiment of the invention is a diagnostic test kit for Alzheimer's disease comprising anti-phospho-MAPK protein antibody and bradykinin. An embodiment provides a method for selecting medication for an Alzheimer's patient comprising selecting a possible therapeutic compound, administering the possible therapeutic compound to the patient, and thereafter, detecting the presence or absence of an abnormally elevated level of a phosphorylated indicator protein in cells of the patient after activating the cells with a compound that stimulates phosphorylation of the indicator protein, the presence of such an elevated level indicating that the possible therapeutic compound is not effective for the patient, and the absence of such a level indicating that the possible therapeutic compound is therapeutic for the patient. The method may further comprise treating or preventing Alzheimer's disease in the subject by administering to the subject the compound shown to be therapeutic for the patient. The invention provides a method of treating or preventing Alzheimer's disease in a subject comprising administering an effective amount of a medicament that (a) inhibits or prevents abnormally elevated phosphorylation of a MAPK protein in cells of the subject as compared to control cells; and/or (b) inhibits events caused by abnormally elevated phosphorylation of said MAPK protein. The medicament may inhibit Erk1/2 phosphorylation, and may be an inhibitor of protein kinase C activity. src protein tyrosine kinase activity, or the IP-3 receptor. The inhibitor may be selected from the group consisting of BiSM-1, PP 1, and 2ABP. In particular, the invention provides a method of diagnosing Alzheimer's disease in a subject, comprising: (a) contacting skin fibroblast cells from the subject and from a non-Alzheimer's control subject with an effective, phosphorylation-stimulating concentration of bradykinin, (b) measuring the amount of phosphorylated Erk1/2 in the subject's cells at one or more time points selected from the group consisting of 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, by Western blotting using an antibody specific for phospho-Erk1/2; (c) measuring the amount of phosphorylated Erk1/2 in cells from a non-Alzheimer's control subject at the same time point or points as in (b) by Western blotting using an antibody specific for phospho-Erk1/2, wherein the amount of phosphorylated Erk1/3 in steps (b) and (c) is normalized to the amount of protein present in said cells; (d) comparing the amount of phosphorylated Erk1/2 in the subject's cells with the amount of phosphorylated Erk1/2 in the control cells at said time points, wherein an increased amount of phosphorylated Erk1/2 in the subject's cells compared to the control cells at one of more of said time points is diagnostic of Alzheimer's disease. The method may further comprise contacting the subject's cells with one or more inhibitors selected from the group consisting of the inhibitor of protein kinase C activity, BiSM-1, the inhibitor of C-src protein tyrosine kinase activity, PP1; and the inhibitor of the IP-3 receptor, 2-aminoethoxydiphenyl borate, wherein the bradykinin-induced increase in the amount of phosphorylated Erk1/2 in the subject's cells compared to the control cells is reduced by said inhibitor. A further embodiment of the invention provides a method for screening compounds to identify a compound useful for treatment or prevention of Alzheimer's disease comprising: (a) contacting test skin fibroblasts from an AD subject with a compound being screened; (b) contacting control skin fibroblasts from said subject with a control agent for said compound or incubating said control fibroblasts in the absence or either said compound or said control agent; (c) before, during, or after step (a) and (b) stimulating the test and the control fibroblasts with an effective, phosphorylation-stimulating concentration of bradykinin, (d) measuring the amount of phosphorylated Erk1/2 in the test and in the control fibroblasts at one or more time points selected from the group consisting of 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, by Western blotting using an antibody specific for phospho-Erk1/2, wherein the amount of phosphorylated Erk1/3 is normalized to the amount of protein present in said test and control fibroblasts; (e) comparing the amount of phosphorylated Erk1/2 in the test fibroblasts with the amount of phosphorylated Erk1/2 in the control fibroblasts, to determine whether the compound inhibits or prevents bradykinin-induced increase in phosphorylation of Erk1/2 in the test cells compared to the control cells, wherein a compound that inhibits or prevents the increased phosphorylation is identified as useful for the treatment of prevention of Alzheimer's disease. A method of the invention is a method of reducing proteolysis of amyloid precursor protein, secretion of amyloid protein β, and/or phosphorylation of tau protein in a human cell, the cell having increased IP3 receptor mediated phosphorylation of MAPK protein compared to a control human cell, comprising contacting the cell with an inhibitor of phosphorylation of MAPK effective to reduce phosphorylation to the level in the control cell The elements of the invention recited herein may be combined or eliminated among the particular embodiments described, as would be apparent to a person of ordinary skill. |
Novel Genes Encoding Novel Proteolytic Enzymes |
The invention relates to newly identified gene sequences that encode novel proteases obtainable from Aspergillus niger. The invention features the full length gene sequence of the novel genes, their cDNA sequences as well as the full-length functional protein and fragments thereof. The invention also relates to methods of using these enzymes in industrial processes and methods of diagnosing fungal infections. Also included in the invention are cells transformed with DNA according to the invention and cells wherein a protease according to the invention is genetically modified to enhance or reduce its activity and/or level of expression. |
1. An isolated polynucleotide that encodes a protease wherein said polynucleotide hybridizes to a polynucleotide of SEQ ID NO: 1 to SEQ ID NO: 57 or the complement thereof or of SEQ ID NO: 58 to SEQ ID NO: 114 or the complement thereof under wash conditions of 1×SSC, 0-1% SDS at 50° C. 2. An isolated polynucleotide according to claim 1, which thus hybridizes under stringent conditions. 3. An isolated polynucleotide according to claim 1 obtainable from a filamentous fungus. 4. An isolated polynucleotide according to claim 3 obtainable from A. niger. 5. An isolated polynucleotide encoding a polypeptide having protease activity, said protein comprising an amino acid sequence of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof. 6. The isolated polynucleotide of claim 5 that encodes a protease or functional portion thereof at least 60% identical to SEQ ID NO: 115 to SEQ ID NO: 171 or to the corresponding functional portion thereof. 7. (canceled) 8. The isolated polynucleotide of claim 1 that has the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 57 or their complements or of SEQ ID NO: 58 to SEQ ID NO: 114 or their complements. 9. A vector comprising a polynucleotide sequence according to claim 1. 10. A vector according to claim 9 wherein said polynucleotide sequence is operatively linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell. 11. A vector according to claim 10 wherein said suitable host cell is a filamentous fungus. 12. A method to prepare a protease comprising the steps of culturing a host cell comprising the vector of claim 10 and isolating said protease from said host cell. 13. An isolated polypeptide having protease activity that has the amino acid sequence of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof. 14. An isolated polypeptide according to claim 13 obtainable from Aspergillus niger. 15. An isolated polypeptide having protein activity obtainable by expressing a vector according to claim 10 in a host cell. 16. A recombinant protease comprising a functional domain of a protease polypeptide, which has an amino acid sequence at least 60% identical to a functional domain of SEQ ID NO: 115 to SEQ ID NO: 171. 17. (canceled) 18. A recombinant host cell comprising a vector according to claim 10. 19. A recombinant host cell expressing a polypeptide according to claim 16. 20. A recombinant host cell comprising a polynucleotide encoding a functionally inactivated protease polypeptide. 21. A recombinant host cell wherein a polynucleotide encoding a protease polypeptide has at least partially been deleted. 22. A recombinant host cell according to claim 18 wherein said host cell is from an Aspergillus species. 23. A recombinant host cell functionally deficient in a protease obtainable by a method comprising said steps of: a. in vitro mutagenesis of a polynucleotide according to claim 1, b. transforming a host cell comprising an endogenous gene comprising a polynucleotide sequence hybridisable to said mutagenised polynucleotide obtained in step a), c. selecting and isolating recombinant host cells in which said endogenous gene is replaced by a mutagenised polynucleotide obtained in step a). 24. Purified antibodies reactive with a polypeptide according to claim 16. 25. Fusion protein comprising a polypeptide sequence according to claim 16. 26. Method for diagnosing whether an organism is infected with Aspergillus comprising said steps of: a. isolating a biological sample from said organism suspected to be infected with Aspergillus, b. isolating nucleic acid from that sample, c. determining whether said isolated nucleic acid comprises polynucleotides hybridisable to a polynucleotide according to claim 1. 27. Method according to claim 26 wherein step c) additionally comprises amplifying said isolated nucleic acid. 28. Method for diagnosing whether an organism is infected with Aspergillus comprising said steps of: a. isolating a biological sample from said organism suspected to be infected with Aspergillus, b. reacting said biological sample with an antibody according to claim 24, c. determining whether immune complexes are formed. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Proteolytic Enzymes Proteins can be regarded hetero-polymers that consist of amino acid building blocks connected by a peptide bond. The repetitive unit in proteins is the central alpha carbon atom with an amino group and a carboxyl group. Except for glycine, a so-called amino acid side chain substitutes one of the two remaining alpha carbon hydrogen atoms. The amino acid side chain renders the central alpha carbon asymmetric. In general, in proteins the L-enantiomer of the amino acid is found. The following terms describe the various types of polymerized amino acids. Peptides are short chains of amino acid residues with defined sequence. Although there is not really a maximum to the number of residues, the term usually indicates a chain which properties are mainly determined by its amino acid composition and which does not have a fixed three-dimensional conformation. The term polypeptide is usually used for the longer chains, usually of defined sequence and length and in principle of the appropriate length to fold into a three-dimensional structure. Protein is reserved for polypeptides that occur naturally and exhibit a defined three-dimensional structure. In case the proteins main function is to catalyze a chemical reaction it usually is called an enzyme. Proteases are the enzymes that catalyze the hydrolysis of the peptide bond in (poly)peptides and proteins. Under physiological conditions proteases catalyse the hydrolysis of the peptide bond. The International Union of Biochemistry and Molecular Biology (1984) has recommended to use the term peptidase for the subset of peptide bond hydrolases (Subclass E.C 3.4.). The terms protease and peptide hydrolase are synonymous with peptidase and may also be used here. Proteases comprise two classes of enzymes: the endo-peptidases and the exo-peptidases, which cleave peptide bonds at points within the protein and remove amino acids sequentially from either N or C-terminus respectively. Proteinase is used as a synonym for endo-peptidase. The peptide bond may occur in the context of di-, tri-, tetra-peptides, peptides, polypeptides or proteins. In general the amino acid composition of natural peptides and polypeptides comprises 20 different amino acids, which exhibit the L-configuration (except for glycine which does not have a chiral centre). However the proteolytic activity of proteases is not limited to peptides that contain only the 20 natural amino acids. Peptide bonds between so-called non-natural amino acids can be cleaved too, as well as peptide bonds between modified amino acids or amino acid analogues. Some proteases do accept D enantiomers of amino acids at certain positions. In general the remarkable stereoselectivity of proteases makes them very useful in the process of chemical resolution. Many proteases exhibit interesting side activities such as esterase activity, thiol esterase activity and (de)amidase activity. These side activities are usually not limited to amino acids only and might turn out to be very useful in bioconversions in the area of fine chemicals. There are a number of reasons why proteases of filamentous fungi, eukaryotic microorganisms, are of particular interest. The basic process of hydrolytic cleavage of peptide bonds in proteins appears costly and potentially detrimental to an organism if not properly controlled. The desired limits to proteolytic action are achieved through the specificity of proteinases, by compartmentalization of proteases and substrates within the cell, through modification of the substrates allowing recognition by the respective proteases, by regulation via zymogen activation, and the presence or absence of specific inhibitors, as well through the regulation of protease gene expression. In fungi, proteases are also involved in other fundamental cellular processes, including intracellular protein turnover, processing, translocation, sporulation, germination and differentiation. In fact, Aspergillus nidulans and Neurospora crassa have been used as model organisms for analyzing the molecular basis of a range of physiological and developmental processes. Their genetics enable direct access to biochemical and genetical studies, under defined nutrient and cultivation conditions. Furthermore, a large group of fungi pathogenic to humans, live-stock and crop, has been isolated and proteolysis has been suggested to play a role in their pathogenicity (host penetration, countering host defense mechanisms and/or nutrition during infection). Proteases are also frequently used in laboratory, clinical and industrial processes; both microbial and non-microbial proteases are widely used in the food industry (baking, brewing, cheese manufacturing, meat tenderizing), in tanning industry and in the manufacture of biological detergents (Aunstrup, 1980). The commercial interest in exploiting certain filamentous fungi, especially the Aspergilli, as hosts for the production of both homologous and heterologous proteins, has also recently renewed interests in fungal proteases (van Brunt, 1986ab). Proteases often cause problems in heterologous expression and homologous overexpression of proteins in fungi. In particular, heterologous expression is hampered by the proteolytic degradation of the expressed products by homologous proteases. These commercial interests have resulted in detailed studies of proteolytic spectra and construction of protease deficient strains and have improved the knowledge about protease expression and regulation in these organisms. Consequently there is a great need to identify and eliminate novel proteases in filamentous fungi. Micro-organisms such as for example fungi are particularly useful in the large scale production of proteins. In particular when such proteins are secreted into the medium. Proteolytic enzymes play a role in these production processes. On the one hand particular proteolytic enzymes are in general required for proper processing of the target protein and the metabolic well-being of the production host. On the other hand proteolytic degradation may significantly decrease the yield of secreted proteins. Poor folding in the secretion pathway may lead to degradation by intracellular proteases. This might be a particular problem with producing heterologous proteins. The details of the proteolytic processes, which are responsible for the degradation of the proteins that are diverted from the secretory process in fungi are not exactly known. In eukaryotes the degradation of cellular proteins is achieved by a proteasome and usually involves ubiquitin labelling of proteins to be degraded. In fungi, proteasomal and vacuolar proteases are also likely candidates for the proteolytic degradation of poorly folded secretory proteins. The proteolytic degradation is likely cytoplasmic, but endoplamatic reticulum resident proteases cannot be excluded. From the aspect of production host strain improvement the proteolytic system may be an interesting target for genetic engineering and production strain improvement. Additional copies of protease genes, over-expression of certain proteases, modification of transcriptional control, as well as knock out procedures for deletion of protease genes may provide a more detailed insight in the function a given protease. Deletion of protease encoding genes can be a valuable strategy for host strain improvement in order to improve production yield for homologous as well as heterologous proteins. Eukaryotic microbial proteases have been reviewed by North (1982). More recently, Suarez Rendueles and Wolf (1988) have reviewed the S. cerevisiae proteases and their function. Apart from the hydrolytic cleavage of bonds, proteases may also be applied in the formation of bonds. Bonds in this aspect comprise not only peptide and amide bonds but also ester bonds. Whether a protease catalyses the cleavage or the formation of a particular bond does in the first place depend on the thermodynamics of the reaction. An enzyme such as a protease does not affect the equilibrium of the reaction. The equilibrium is dependent on the particular conditions under which the reaction occurs. Under physiological conditions the thermodynamics of the reactions is in favour of the hydrolysis of the peptide due to the thermodynamically very stable structure of the zwitterionic product. By application of physical-chemical principles to influence the equilibrium, or by manipulating the concentrations or the nature of the reactants and products, or by exploiting the kinetic parameters of the enzyme reaction it is possible to apply proteases for the purpose of synthesis of peptide bonds. The addition of water miscible organic solvents decreases the extent of ionisation of the carboxyl component, thereby increasing the concentration of substrate available for the reaction. Biphasic systems, water mimetics, reverse micelles, anhydrous media, or modified amino and carboxyl groups to invoke precipitation of products are often employed to improve yields. When the proteases with the right properties are available the application of proteases for synthesis offers substantial advantages. As proteases are stereoselective as well as regio-selective, sensitive groups on the reactants do usually not need protection and reactants do not need to be optically pure. As conditions of enzymatic synthesis are mild, racemization and decomposition of labile reactants or products can be prevented. Apart from bonds between amino acids, also other compounds exhibiting a primary amino group, a thiol group or a carboxyl group may be linked by properly selected proteases. In addition esters, thiol esters and amides may be synthesized by certain proteases. Protease have been shown to exhibit regioselectively in the acylation of mono, di- and tri-saccharides, nucleosides, and riboflavin. Problems with stability under the sometimes harsh reaction conditions may be prevented by proper formulation. Encapsulation and immobilisation do not only stabilise enzymes but also allow easy recovery and separation from the reaction medium. Extensive crosslinking, treatment with aldehydes or covering the surface with certain polymers such as dextrans, polyethyleneglycol, polyimines may substantially extend the lifetime of the biocatalyst. The Natural Roles of Proteases Traditionally, proteases have been regarded as degrading enzymes, capable of cleaving proteins into small peptides and/or amino acids, and whose role it is to digest nutrient protein or to participate in the turnover of cellular proteins. In addition, it has been shown that proteases also play key roles in a wide range of cellular processes, via mechanisms of selective modification by limited proteolysis, and thus can have essential regulatory functions (Holzer and Tschensche 1979; Holzer and Heinrich, 1980). The specificity of a proteinase is assumed to be closely related to its physiological function and its mode of expression. With respect to the function of a particular protease, its localisation is often very important; for example, a lot of the vacuolar and periplasmic proteases are involved in protein degradation, while many of the membrane-bound proteases are important in protein processing (Suarez Rendueles and Wolf, 1988). The different roles of proteases in many cellular processes can be divided into four main functions of proteases: 1) protein degradation, 2) posttranslational processing and (in)activation of specific proteins, 3) morphogenesis, and 4) pathogenesis. An obvious role for proteases in organisms which utilise protein as a nutrient source is in the hydrolysis of nutrients. In fungi, this would involve the degradation outside the cells by extracellular broad specificity proteases. Protein degradation is also important for rapid turnover of cellular proteins and allows the cell to remove abnormal proteins and to adapt their complement of protein to changing physiological conditions. Generally, proteases of rather broad specificity should be extremely well-controlled in order to protect the cell from random degradation of other than correct target proteins. Contrary to the hydrolysis the synthesis of polypeptides occurs in vivo by an ATP driven process on the ribosome. Ultimately the sequence in which the amino acids are linked is dictated by the information derived from the genome. This process is known as the transcription. Primary translation products are often longer than the final functional products, and after the transcription usually further processing of such precursor proteins by proteases is required. Proteases play a key role in the maturation of such precursor proteins to obtain the final functional protein. In contrast to the very controlled trimming and reshaping of proteins, proteases can also be very destructive and may completely degrade polypeptides into peptides and amino acids. In order to avoid that proteolytic activity is unleashed before it is required, proteases are subject to extensive regulation. Many proteases are synthesized as larger precursors known as zymogens, which become activated when required. Remarkably this activation always occurs by proteolysis. Apart from direct involvement in the processing, selective activation and inactivation of individual proteins are well-known phenomena catalyzed by specific proteases. The selectivety of limited proteolysis appears to reside more directly in the proteinase-substrate interaction. Specificity may be derived from the proteolytic enzyme which recognizes only specific amino acid target sequences. On the other hand, it may also be the result of selective exposure of the ‘processing site’ under certain conditions such as pH, ionic strength or secondary modifications, thus allowing an otherwise non-specific protease to catalyze a highly specific event. The activation of vacuolar zymogens by limited proteolysis gives an example of the latter kind. Morphogenesis or differentiation can be defined as a regulated series of events leading to changes from one state to another in an organism. Although direct relationships between proteases and morphological effects could not be established in many cases, the present evidence suggests a significant involvement of proteases in fungal morphogenesis; apart form the observed extensive protein turnover during differentiation, sporulation and spore germination, proteases are thought to be directly involved in normal processes as hyphal tip branching and septum formation, (Deshpande, 1992). Species of Aspergillus , in particular A. fumigatus and A. flavus , have been implicated as the causative agents of a number of diseases in humans and animals called aspergillosis (Bodey and Vartivarian, 1989). It has been repeatedly suggested that proteases are involved in virulence of A. fumigatus and A. flavus like there are many studies linking secreted proteases and virulence of bacteria. In fact, most human infections due to Aspergillus species are characterised by an extensive degradation of the parenchyma of the lung which is mainly composed of collagen and elastin (Campbell et al., 1994). Research has been focussed on the putative role of the secreted proteases in virulence of A. fumigatus and A. flavus which are the main human pathogens and are known to possess elastinolytic and collagenic activities (Kolattukudy et al., 1993). These elastinolytic activities were shown to correlate in vitro with infectivity in mice (Kothary et al., 1984). Two secreted proteases are known to be produced by A. fumigatus and A. flavus , an alkaline serine protease (ALP) and a neutral metallo protease (MEP). In A. fumigatus both the genes encoding these proteases were isolated, characterised and disrupted (Reicherd et al., 1990; Tang et al, 1992, 1993; Jaton-Ogay et al., 1994). However, alp mep double mutants showed no differences in pathogenecity when compared with wild type strains. Therefore, it must be concluded that the secreted A. fumigatus proteases identified in vitro are not essential factors for the invasion of tissue (Jaton Ogay et al., 1994). Although A. fumigatus accounts for only a small proportion of the airborne mould spores, it is the most frequently isolated fungus from lung and sputem (Schmitt et al., 1991). Other explanations for the virulence of the fungus could be that the conditions in the bronchia (temperature and nutrients) are favourable for the parasitic growth of A. fumigatus . As a consequence, invasive apergillosis could be a circumstancial event, when the host pathogenic defences have been weakened by immunosuppressive treatments or diseases like AIDS. Four major classes of proteases are known and are designated by the principal functional groups in their active site: the ‘serine’, the ‘thiol’ or ‘cysteine’, the ‘aspartic’ or ‘carboxyl’ and the ‘metallo’ proteases. A detailed state of the art review on these major classes of proteases, minor classes and unclassified proteases can be found in Methods in Enzymology part 244 and 248 (A. J. Barrett ed, 1994 and 1995). Specificity of Proteases Apart from the catalytic machinery of proteases another important aspect of proteolytic enzymes is the specificity of proteases. The specificity of a protease indicates which substrates the protease is likely to hydrolyze. The twenty natural amino acids offer a large number of possibilities to make up peptides. Eg with twenty amino acids one can make up already 400 dipeptides and 800 different tripeptide, and so on. With longer peptides the number of possibilities will become almost unlimited. Certain proteases hydrolyze only particular sequences at a very specific position. The interaction of the protease with the peptide substrate may encompass one up to ten amino acid residues of the peptide substrate. With large proteinacious substrates there may be even more residues of the substrate that interact with the proteases. However this likely involves less specific interactions with protease residues outside the active site binding cleft. In general the specific recognition is restricted to the linear peptide, which is bound in the active site of the protease. The nomenclature to describe the interaction of a substrate with a protease has been introduced in 1967 by Schechter and Berger (Biochem. Biophys. Res. Corn., 1967, 27, 157-162) and is now widely used in the literature. In this system, it is considered that the amino acid residues of the polypeptide substrate bind to so-called sub-sites in the active site. By convention, these sub-sites on the protease are called S (for sub-sites) and the corresponding amino acid residues are called P (for peptide). The amino acid residues of the N-terminal side of the scissile bond are numbered P3, P2, P1 and those residues of the C-terminal side are numbered P1′, P2′, P3′. The P1 or P1′ residues are the amino acid residues located near the scissile bond. The substrate residues around the cleavage site can then be numbered up to P8. The corresponding sub-sites on the protease that complement the substrate binding residues are numbered S3, S2, S1, S1′, S2′, S3′, etc, etc. The preferences of the sub-sites in the peptide binding site determine the preference of the protease for cleaving certain specific amino acid sequences at a particular spot. The amino acid sequence of the substrate should conform with the preferences exhibited by the sub-sites. The specificity towards a certain substrate is clearly dependant both on the binding affinity for the substrate and on the velocity at which subsequently the scissile bond is hydrolysed. Therefore the specificity of a protease for a certain substrate is usually indicated by its kcat/Km ratio, better known as the specificity constant. In this specificity constant kcat represents the turn-over rate and Km is the dissociation constant. Apart from amino acid residues involved in catalysis and binding, proteases contain many other essential amino acid residues. Some residues are critical in folding, some residues maintain the overall three dimensional architecture of the protease, some residues may be involved in regulation of the proteolytic activity and some residue may target the protease for a particular location. Many proteases contain outside the active site one or more binding sites for metal ions. These metal ions often play a role in stabilizing the structure. In addition secreted eukaryotic microbial proteases may be extensively glycosylated. Both N- and O-linked glycosylation occurs. Glycosylation may aid protein folding, may increase solubility, prevent aggregation and as such stabilize the mature protein. In addition the extent of glycosylation may influence secretion as well as water binding by the protein. Regulation of Proteolytic Activity A substantial number of proteases are subject to extensive regulation of the proteolytic activity in order to avoid undesired proteolytic damage. To a certain extent this regulation takes place at transcription level. For example in fungi the transcription of secreted protease genes appears to be sensitive to external carbon and nitrogen sources, whereas genes encoding intracellular proteases are insensitive. The extracellular pH is sensed by fungi and some genes are regulated by pH. In this process transcriptional regulator proteins play a crucial role. Proteolytic processing of such regulator proteins is often the switch that turns the regulator proteins either on or off. Proteases are subject to intra- as well as intermolecular regulation. This implies certain amino acids in the proteolytic enzyme molecule that are essential for such regulation. Proteases are typically synthesized as larger precursors known as zymogens, which are catalytically inactive. Usually the peptide chain extension rendering the precursor protease inactive is located at the amino terminus of the protease. The precursor is better known as pro-protein. As many of the proteases processed in this way are secreted from the cells they contain in addition a signal sequence (pre sequence) so that the complete precursor is synthesized as a pre-pro-protein. Apart from rendering the protease inactive the pro-peptide often is essential for mediating productive folding. Examples of proteases include serine proteases (alpha lytic protease, subtilisin, aqualysin, prohormone convertase), thiol proteases (cathepsin L and cruzian), aspartic proteases (proteinase A and cathepsin D) and metalloproteases. In addition the pro-peptide might play a role in cellular transport either alone or in conjunction with signal peptides. It may facilitate interaction with cellular chaperones or it may facilitate transport over the membrane. The size of the extension in the precursor pre-pro-protein may vary substantially, ranging from a short peptide fragment to a polypeptide, which can exist as an autonomous folding unit. In particular these larger extensions are often observed to be strong inhibitors of the protease even after cleavage from the protease. It was observed that even after cleavage such pro-peptides could assist in proper folding of the proteases. As such pro-peptides can be considered to function as molecular chaperones and separate or additional co-expression of such pro-peptides could be advantageous for protease production. There is substantial difference in the level of regulation between proteases that are secreted into the medium and proteases that remain intracellular. Proteases secreted into the medium are usually after activation no longer subject to control and therefore are usually relatively simple in their molecular architecture consisting of one globular module. Intracellular proteases are necessarily subject to continuous control in order to avoid damage to the cells. In contrast with zymogens of secreted proteases in more complex regulatory proteases very large polypeptide segments may be inserted between the signal and the zymogen activation domain of the proteolytic module. Structure-function studies indicate that such non-protease parts may be involved in interactions with macroscopic structures, membranes, cofactors, substrates, effectors, inhibitors, ions, that regulate activity and activation of the proteolytic module(s) or its (their) zymogens. The non-proteolytic modules exhibit remarkable variation in size and structure. Many of the modules can exist as such independently from the proteolytic module. Therefore such modules can be considered to correspond to independent structural and functional units that are autonomous with respect to folding. The value of such a modular organization is that acquisition of new modules can endow the recipient protease with new novel binding specificities and can lead to dramatic changes in its activity, regulation and targeting. The principle of modular organized proteolytic enzymes may also be exploited by applying molecular biology tools in order to create novel interactions, regulation, specificity, and/or targeting by shuffling of modules. Although in general such additional modules are observed as N or C terminal extension, also large insertions within the exterior loops of the catalytic domain have been observed. It is believed that also in this case the principal fold of the protease represents still the essential topology to form a functional proteolytic entity and that the insertion can be regarded as substructure folded onto the surface of the proteolytic module. Molecular Structure In principle the modular organization of larger proteins is a general theme in nature. In particular within the larger multimodular frameworks typical proteolytic modules show sizes of 100 to 400 amino acids on the average. This corresponds with the average size of most of the globular proteolytic enzymes that are secreted into the medium. As discussed above polypeptide modules are polypeptide fragments, which can fold and function as independent entities. Another term for such modules is domains. However domain is used in a broader context than module. The term domain as used herein refers usually to a part of the polypeptide chain that depicts in the three-dimensional structure a typical folding topology. In a protein domains interact to varying extents, but less extensively than do the structural elements within domains. Other terms such as subdomain and folding unit are also used in literature. As such it is observed that many proteins that share a particular functionality may share the same domains. Such domains can be recognized from the primary structure that may show certain sequence patterns, which are typical for a particular domain. Typical examples are the mononucleotide binding fold, cellulose binding domains, helix-turn-helix DNA binding motif, zinc fingers, EF hands, membrane anchors. Modules refer to those domains which are expected to be able to fold and function autonomously. A person skilled in the art knows how to identify particular domains in a primary structure by applying commonly available computer software to said structure and homologous sequences from other organisms or species. Although multimodular or multidomain proteins may appear as a string of beads, assemblies of substantial more complex architecture have been observed. In case the various beads reside on the same polypeptide chain the beads are generally called modules or domains. When the beads do not reside on one and same polypeptide chain but form assemblies via non-covalent interactions then the term subunit is used to designate the bead. Subunits may be transcribed by one and the same gene or by different genes. The multi-modular protein may become proteolytically processed after transcription leading to multiple subunits. Individual subunits may consist of multiple domains. Typically the smaller globular proteins of 100-300 amino acids usually consist only of one domain. Molecular Classification of Proteolytic Enzymes In general proteases are classified according to their molecular properties or according to their functional properties. The molecular classification is based on the primary structure of the protease. The primary structure of a protein represents its amino acid sequence, which can be derived from the nucleotide sequence of the corresponding gene. Tracing extensively the similarities in the primary structures may allow for the notice of similarities in catalytic mechanism and other properties, which even may extend to functional properties. The term family is used to describe a group of proteases that show evolutionary relationship based on similarity between their primary structures. The members of such a family are believed to have arisen by divergent evolution from the same ancestor. Within a family further sub-grouping of the primary structures based on more detailed refinement of sequence comparisons results in subfamilies. Classification according to three-dimensional fold of the proteases may comprise secondary structure, tertiary structure and quarternary structure. In general the classification on secondary structure is limited to content and gross orientation of secondary structure elements. Similarities in tertiary structure have led to the recognition of superfamilies or clans. A superfamily or a clan is a group of families that are thought to have common ancestry as they show a common 3-dimensional fold. In general tertiary structure is more conserved than the primary structure. As a consequence similarity of the primary structure does not always reflect similar functional properties. In fact functional properties may have diverged substantially resulting in interesting new properties. At present quarternary structure has not been applied to classify various proteases. This might be due to a certain bias of the structural databases towards simple globular proteases. Many proteolytic systems that are subject to activation, regulation, or complex reaction cascades are likely to consist of multiple domains or subunits. General themes in the structural organization of such protease systems may lead to new types of classification. Classification According to Specificity. In absence of sequence information proteases haven been subject to various type of functional classification. The classification and naming of enzymes by reference to the reactions which are catalyzed is a general principle in enzyme nomenclature. This approach is also the underlying principle of the EC numbering of enzymes ( Enzyme Nomenclature 1992 Academic Press, Orlando). Two types of proteases (EC 3.4) can be recognized within Enzyme Nomenclature 1992, those of the exo-peptidases (EC 3.4.11-19) and those of the endo-peptidases (EC 3.4.21-24, 3.4.99). Endo-peptidases cleave peptide bonds in the inner regions of the peptide chain, away from the termini. Exo-peptidases cleave only residues from the ends of the peptide chain. The exo-peptidases acting at the free N-terminus may liberate a single amino acid residue, a dipeptide or a tripeptide and are called respectively amino peptidases (EC 3.4.11), dipeptidyl peptidases (EC 3.4.14) and tripeptidyl peptidase (EC 3.3.14). Proteases starting peptide processing from the carboxyl terminus liberating a single amino acid are called carboxy peptidase (EC 3.4.16-18). Peptidyl-dipeptidases (EC 3.4.15) remove a dipeptide from the carboxyl terminus. Exo- and endo-peptidase in one are the dipeptidases (EC 3.4.13), which cleave specifically only dipeptides in their two amino acid halves. Omega peptidases (EC 3.4.19) remove terminal residues that are either substituted, cyclic, or linked by isopeptide bonds Apart from the position where the protease cleaves a peptide chain, for each type of protease a further division is possible based on the nature of the preferred amino acid residues in the substrate. In general one can distinguish proteases with broad, medium and narrow specificity. Some proteases are simply named after the specific proteins or polypeptides that they hydrolyze, e.g. keratinase, collagenase, elastase. A narrow specificity may pin down to one particular amino acid or one particular sequence which is removed or which is cleaved respectively. When the protease shows a particular preference for one aminoacid in the P1 or P1′ position the name of this amino acid may be a qualifier. For example prolyl amino peptidase removes proline from the amino terminus of a peptide (proline is the P1 residue). X-Pro or proline is used when the bond on the imino side of the proline is cleaved (proline is P1′ residue), eg proline carboxypeptidase removes proline from the carboxyl terminus. Prolyl endopeptidase (or Pro-X) cleaves behind proline while proline endopeptidase (X-Pro) cleaves in front of a proline. Amino acid residue in front of the scissile peptide bond refers to the amino acid residue that contributes the carboxyl group to the peptide bond. The amino acids residue behind the scissile peptide bond refers to the amino acid residue that contributes the amino group to the peptide bond. According to the general convention an amino acid chain runs from amino terminus (the start) to the carboxyl terminus (the end) and is numbered accordingly. Endo proteases may also show clear preference for a particular amino acid in the P1 or P1′ position, eg glycyl endopeptidase, peptidyl-lysine endopeptidase, glutamyl endopeptidase. In addition proteases may show a preference for a certain group of amino acids that share a certain resemblance. Such a group of preferred amino acids may comprise the hydrophobic amino acids, only the bulky hydrophobic amino acids, small hydrophobic, or just small amino acids, large positively charged amino acids, etc, etc. Apart from preferences for P1 and P1′ residues also particular preferences or exclusions may exist for residues preferred by other subsites on the protease. Such multiple preferences can result in proteases that are very specific for only those sequences that satisfy multiple binding requirements at the same time. In general it should be realized that protease are rather promiscuous enzymes. Even very specific protease may cleave peptides that do not comply with the generally observed preference of the protease. In addition it should be realized that environmental conditions such as pH, temperature, ionic strength, water activity, presence of solvents, presence of competing substrates or inhibitors may influence the preferences of the proteases. Environmental condition may not only influence the protease but also influence the way the proteinacious substrate is presented to the protease. Classification by Catalytic Mechanism. Proteases can be subdivided on the basis of their catalytic mechanism. It should be understood that for each catalytic mechanism the above classification based on specificity leads to further subdivision for each type of mechanism. Four major classes of proteases are known and are designated by the principal functional group in the active site: the serine proteases (EC 3.4.21 endo peptidase, EC 3.4.16 carboxy peptidase), the thiol or cysteine proteases (EC 3.4.22 endo peptidase, EC 3.4.18 carboxy peptidase), the carboxyl or aspartic proteases (EC 3.4.23 endo peptidase) and metallo proteases (EC 3.4.24 endo peptidase, EC 3.4.18 carboxy peptidase). There are characteristic inhibitors of the members of each catalytic type of protease. These small inhibitors irreversibly modify an amino acid residue of the protease active site. For example, the serine protease are inactivated by Phenyl Methane Sulfonyl Fluoride (PMSF) and Diisopropyl Fluoro Phosphate (DFP), which react with the active Serine whereas the chloromethylketone derivatives react with the Histidine of the catalytic triad. Phosphoramidon and 1,10 Phenanthroline typically inhibit metallo proteases. Inhibition by Pepstatin generally indicates an aspartic protease. E64 inhibits thiol protease specifically. Amastatin and Bestatin inhibit various aminopeptidases. Substantial variations in susceptibility of the proteases to the inhibitors are observed, even within one catalytic class. To a certain extent this might be related to the specificity of the protease. In case binding site architecture prevents a mechanism based inhibitor to approach the catalytic site, then such a protease escapes from inhibition and identification of the type of mechanism based on inhibition is prohibited. Chymostation for example is a potent inhibitor for serine protease with chymotrypsin like specificity, Elastatinal inhibits elastase like serine proteases and does not react with trypsin or chymostrypsin, 4 amido PMSF (APMSF) inhibits only serine proteases with trypsin like specificity. Extensive accounts of the use of inhibitors in the classification of proteases include Barret and Salvesen, Proteinase Inhibitors , Elsevier Amstardam, 1986; Bond and Beynon (eds), Proteolytic Enzymes, A Practical Approach , IRL Press, Oxford, 1989; Methods in Enzymology, eds E. J. Barret, volume 244, 1994 and volume 248, 1995; E. Shaw, Cysteinyl proteinases and their selective inactivation , Adv Enzymol. 63:271-347 (1990) Classification According to Optimal Performance Conditions. The catalytic mechanism of a proteases and the requirement for its conformational integrity determine mainly the conditions under which the protease can be utilized. Finding the protease that performs optimal under application conditions is a major challenge. Often conditions at which proteases have to perform are not optimal and do represent a compromise between the ideal conditions for a particular application and the conditions which would suit the protease best. Apart from the particular properties of the protease it should be realized that also the presentation of a proteinacious substrates is dependant on the conditions, and as such determines also which conditions are most effective for proteolysis. Specifications for the enzyme that are relevant for application comprise for example the pH dependence, the temperature dependence, sensitivity for or the dependence of metal ions, ionic strength, salt concentration, solvent compatibility. Another factor of major importance is the specific activity of a protease. The higher the enzyme's specific activity, the less enzyme is needed for a specific conversion. Lower enzyme requirements imply lower costs and lower protein contamination levels. The pH is a major parameter that determines protease performance in an application. Therefor pH dependence is an important parameter to group proteases. The major groups that are recognized are the acid proteases, the neutral proteases, the alkaline proteases and the high alkaline proteases. The optimum pH matches only to some extent the proteolytic mechanism, eg aspartic protease show often an optimum at acidic pH, metalloproteases and thiol proteases often perform optimal around neutral pH to slightly alkaline, serine peptidases are mainly active in the alkaline and high alkaline region. For each class exceptions are known. In addition the overall water activity of the system plays a role. The pH optimum of a protease is defined as the pH range where the protease exhibits an optimal hydrolysis rate for the majority of its substrates in a particular environment under particular conditions. This range can be narrow, e.g. one pH unit, as well as quite broad, 3-4 pH units. In general the pH optimum is also dependant on the nature of the proteinacious substrate. Both the turnover rate as well as the specificity may vary as a function of pH. For a certain efficacy it can be desirable to use the protease far from its pH optimum because production of less desired peptides is avoided. Less desired peptides might be for example very short peptides or peptides causing a bitter taste. In addition a more narrow specificity can be a reason to choose conditions that deviate from optimal conditions with respect to turnover rate. Dependant on the pH the specificity may be narrow, e.g. only cleaving the peptide chain in one particular position or before or after one particular amino acid, or broader, e.g. cleaving a chain at multiple positions or cleaving before or after more different types of amino acids. In fact the pH dependence might be an important tool to regulate the proteolytic activity in an application. In case the pH shifts during the process the proteolysis might cease spontaneously without the need for further treatment to inactivate the protease. In some cases the proteolysis itself may be the driver of the pH shift. Very crucial for application of proteases is their handling and operating stability. As protease stability is strongly affected by the working temperature, stability is often also referred to as thermostability. In general the stability of a protease indicates how long a protease retains its proteolytic activity under particular conditions. Particular conditions may comprise fermentation conditions, conditions during isolation and down stream processing of the enzyme, storage conditions, formulation and operating or application conditions. In case particular conditions encompass elevated temperatures stability in general refers to thermostability. Apart from the general causes for enzyme inactivation such as chemical modification, unfolding, aggregation etc, main problem with proteases is that they are easy subject to autodegradation. Especially for the utilization of proteases the temperature optimum is a relevant criterion to group proteases. Although there are different definitions, economically the most useful definition is the temperature or the temperature range in which the protease is most productive in a certain application. Protease productivity is a function of both the stability and the turnover rate. Where elevated temperature in general will increase the turnover rate, rapid inactivation will counteract the increase in turnover rate and ultimately lead to low productivity. The conformational stability of the protease under a given process condition will determine its maximum operating temperature. The temperature at which the protease looses it active conformation, often indicated as unfolding or melting point, can be determined according various methods, for example NMR, Circular Dichroism Spectroscopy, Differential Scanning Calorimetry etc etc. For protease unfolding is usually accompanied by a tremendous increase in autodegradation rate. In applications where low temperatures are required protease may be selected with emphasis on a high intrinsic activity at low to moderate temperature. As under such conditions inactivation is relatively slow, under these conditions activity might largely determine productivity. In processes where only during a short period protease activity is required, the stability of the protease might be used as a switch to turn the protease off. In such case more labile instead of very thermostable protease might be preferred. Other environmental parameters which may play a role in selecting the appropriate protease may be its sensitivity to salts. The compatibility with metal ions which are found frequently at low concentrations in various natural materials can be crucial for certain applications. In particular with metallo proteases certain ions may replace the catalytic metal ion and reduce or even abolish activity completely. In some applications metal ions have to be added on purpose in order to prevent the washout of the metal ions coordinated to the protease. It is well known that for the sake of enzyme stability and life-time, calcium ions have to be supplied in order to prevent dissociation of protein bound calcium. Most microorganisms show a certain tolerance with respect to adapting to changes in the environmental condition. As a consequence at least the proteolytic spectrum that the organism is able to produce are likely to show at least similar tolerances. Such a proteolyitic spectrum might be covered by many proteases covering together the hole spectrum or by only a few proteases of a broad spectrum. Taking into account the whole proteolytic spectrum of a microorganism it can be very important to take the location into account. Cellular Localisation and Characterization of Proteolytic Processing and Degradation From an industrial point of view the proteases which are excreted from the cell have specific advantages with respect to producibility at a large scale and stress tolerance as they have to survive without protection of the cell. The large group of cellular protease can be further subdivided in soluble and membrane bound. Membrane bound may comprise protease at the inside as well the outside of the membrane. Intracellular soluble protease may be subdivided further according to specific compartments of the cell where they do occur. As the cell shields the proteases to some extent from the environment and because the cell controls the conditions in the cell, intracellular protease might be more sensitive to large environmental changes and their optima might correlate better with the specific intacellualr conditions. Knowing the conditions of the cellular department where the protease resides might indicate their preferences. Where extracellular protease in general do not require any regulation any more once excreted from the cell, intracellular proteases are often subject to more complicated control and regulation. With respect to the function of a particular protease, its localisation is often very important; for example, a lot of the vacuolar and periplasmic proteases are involved in protein degradation, while many of the membrane-bound proteases are important in protein processing (Suarez Rendueles and Wolf, 1988). A comprehensive review on the biological properties and evolution of proteases has been published in van den Hombergh: Thesis Landbouwuniversiteit Wageningen: An analysis of the proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2, which is hereby incorporated by reference herein. The Protease Problem An important reason for the interest in microbial proteases are protease related expression problems observed in several expression hosts used in bioprocess industry. The increasing use of heterologous hosts for the production of proteins, by recombinant DNA technology, has recently brought this problem into focus, since it seems that heterologous proteins are more prone to proteolysis (Archer et al., 1992; van den Hombergh et al., 1996b). In S. cerevisiae , already in the early eighties the protease problem and the involvement of several proteases, thus complicating targetted gene disruption approaches to overcome this problem, was recognised. During secretion a protein is exposed to several proteolytic activities residing in the secretory pathway. Additionally, in a prototrophic microorganism as Aspergillus secreted proteins can be exposed to several extracellular proteolytic activities The problem of degradation of heterologously expressed proteins is well documented in Aspergillus (van den Hombergh Thesis Landbouwuniversiteit Wageningen: An analysis of the proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2) and has been reported in the expression of cow prochymosin, human interferon α-2 tPA, GMCSF, IL6, lactoferrin, chicken egg-white lysosyme, porcine pIA2, A. niger pectin lyase B, E. coli enterotoxin B and β-glucoronidase, and Erwinia carotovora pectate lyase 3. The problem of proteolysis may be addressed at several stages in protein production. Bioprocess engineers may address the problem of proteolysis by downstream processing at low temperatures, by early separation of product and protease(s) or by use of protease inhibitors. These may all lead to successful reduction of the problem. However it is certainly not eliminated, because much of the degradation occurs in vivo during the production of the protein. In understanding how proteolysis is controlled in the cell, a major question concerns the recognition mechanism by which proteolysis is triggered. Into what extent are proteolytically susceptable (heterologous) proteins recognised as aberrant because of misfolding or, if correctly folded, as ‘foreign’, because they do not posses features essential for stability which are specific to the host. Various types of stress can cause the overall proteolysis in a cell to increase significantly. Factors known to increase rate of proteolysis include nutrient starvation and various other types of stress (i.e. elevation of temperature, osmotic stress, toxic substances and expression of certain heterologous proteins). To deal with proteolysis-related expression problems in vivo, several approaches have been proven succesfull as will be discussed below. However, we have to keep in mind that true ‘non-proteolytic cells’ cannot exist, since proteolysis by intracellular proteases is involved in many essential metabolic and ‘housekeeping’ reactions. Reducing proteolysis will therefore always be a process in which the changed genetical background which results in decreased proteolytic has to be analysed for potential secundary effects which could lead to reduced protein production (e.g. reduced growth rate or sporulation). Disruption of Proteases in Filamentous Fungal Expression Hosts Berka and coworkers (1990) describe the cloning and disruption of the A. awamori pepA gene. More recently, three disrupted aspartyl proteases in A. niger have been described. Disruptants for both the major extracellular aspartyl proteases and the major vacuolar aspartyl protease were described. Double and triple disruptants were generated via recombination and tested for protease spectra and expression and secretion of the A. niger pectin lyase PELB protein, which is very susceptable to proteolytic degradation (van den Hombergh et al., 1995). Disruption of pepA and pepB resulted both in reduction of extracellular protease activities, 80% and 6%, respectively. In the ΔpepE disruptant also other (vacuolar) protease activities were severely affected caused by inactivating of the proteolytic cascade for other vacuolar proteases. Reduced extracellular activities correlated with reduced in vitro degradation of PELB and improved in vivo expression of pelB (van den Hombergh et al., 1996f). Protease Deficient (prt) Mutants Filamentous Fungi Several Aspergillus protease deficient mutants have been studied whether protein production is improved. Archer and coworkers describe the reduced proteolysis of Hen egg white lysozyme in supernatants of an A. niger double prt mutant generated by Mattern and coworkers (1992) and conclude that although the degradation is not absent, it is significantly reduced. Van den Hombergh et al. (1995) show that the in vitro degradation of A. niger PELB is reduced in all seven prt complementation groups they have isolated. Virtually no degradation is observed in the prtB, prtF and prtG mutants. Recently, the expression of the pelB gene was shown to be improved in six complementation groups tested (prtA-F) and highest expression levels were observed in the prtB, prtF and prtG mutants. In addition to the single mutants, which contained residual extracellular proteolytic activities varying from 2-80% compared to wild type activity, double mutants were generated both by recombination and by additional rounds of mutagenesis. Via this approach several double prt mutants were selected and further characterised, which showed a further reduction of PELB degradation compared to their parental strains. Instead of elimination of protease activities via disruption or mutagenesis, reduced proteolysis can also be achieved via down-regulation of the interfering proteolytic activities. This may be achieved by genetically altering the promoter or other regulatory sequences of the gene. As shown by Fraissinet-Tachet and coworkers (1996) the extracellular proteases in A. niger are all regulated by carbon catabolite repression and nitrogen metabolite repression. Nutrient starvation also causes the overall proteolysis rate in a cell to increase stromgly, which makes sense for a cell that lacks nutrients but posses proteins, that under starvation conditions are not needed or needed only in smaller amounts. In expression strategies which allow high expression on media containing high glucose and ammonium concentrations reduced proteolysis has been reported. Several constitutive glycolytic promoters (gpd and pkiA) are highly expressed under these conditions and can also be used to drive (heterologous) gene expression in continuous fermentations. The type of nutrient starvation imposed can influence different proteases to varying extent, which means that the importance of nutrient conditions in a given process depend on the type of proteolysis that is involved. Specific proteolysis may therefore be induced by conditions of substrate limitation which are frequently used in many large-scale fermentation processes. The protease problem can nowadays be addressed in part by one or more of the above strategies. However, the residual proteolytic activity of yet unidentified proteolytic enzymes still constitutes a major problem in the art. In order to further reduce the level of unwanted proteolysis, there is a great need in the art to identify novel proteases responsible for degradation of homologously and heterologously expressed proteins. This invention provides such novel protease gene sequences encoding novel proteases. Once the primary sequence of a novel protease gene is known, one or more of the above recombinant DNA strategies may be employed to produce (knock-out) mutants with reduced proteolytic activity. Despite the widespread applications of proteases in a great number of industrial processes, current enzymes also have significant shortcomings with respect to at least one of the following properties. When added to animal feed, current proteases are not sufficiently resistant to digestive enzymes present in the gastrointestinal (GI) tract of e.g. pigs and poultry. With respect to another aspect, the currently available enzymes are not sufficiently resistant to specific (high) temperatures and (high) pressure conditions that are applied during extrusion or pelleting operations. Also, the current enzymes are not sufficiently active in a pH range of 3-7, conditions prevailing in many food, beverage products as well as in in the GI tract of most animals. According to yet another aspect the specificity of the currently available proteases is very limited which results in the inability of the existing enzymes to degrade or to dissolve certain “protease resistant” proteins thus resulting in low peptide or amino acid yields. Moreover proteases with new specificities allow the synthesis of new peptides. Yet another drawback of the currently available enzymes is their low specific activity. It is therefore clear that for a large number of applications a strong desire exists for proteases that are more resistant to digestive enzymes, high temperature and/or pressure and which exhibit novel specificities regarding their sites of hydrolysis. The present invention provides such enzymes. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides for novel polynucleotides encoding novel proteases. More in particular, the invention provides for polynucleotides having a nucleotide sequence that hybridises (preferably under highly stringent conditions) to a sequence according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or to a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114. Consequently, the invention provides nucleic acids that are about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114. In a more preferred embodiment the invention provides for such an isolated polynucleotide obtainable from a filamentous fungus, preferably Aspergilli, in particular A. niger is preferred. In one embodiment, the invention provides for an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof. In a further preferred embodiment, the invention provides an isolated polynucleotide encoding at least one functional domain of a polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof. In a preferred embodiment the invention provides a protease gene according to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57. In another aspect the invention provides a polynucleotide, preferably a cDNA encoding an A. niger protease selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or variants or fragments of that polypeptide. In a preferred embodiment the cDNA has a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114 or functional equivalents thereof. A genomic clone encoding a polypeptide according to the invention may also be obtained by selecting suitable probes to specifically amplify a genomic region corresponding to any of the sequences according to SEQ ID NO: 1 to SEQ ID NO: 57 or fragments thereof, hybridising that probe under suitable conditions to genomic DNA obtained from a suitable organism, such as Aspergillus , e.g. A. niger , amplifying the desired fragment e.g. by PCR (polymerase chain reaction) followed by purifying and cloning of the amplified fragment. In an even further preferred embodiment, the invention provides for a polynucleotide comprising the coding sequence of the genomic polynucleotides according to the invention, preferred is a polynucleotide sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114. In another preferred embodiment, the invention provides a cDNA obtainable by cloning and expressing a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 into a suitable host organism, such as A. niger. A polypeptide according to the invention may also be obtained by cloning and expressing a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 into a suitable host organism, such as A. niger. The invention also relates to vectors comprising a polynucleotide sequence according to the invention and primers, probes and fragments that may be used to amplify or detect the DNA according to the invention. In a further preferred embodiment, a vector is provided wherein the polynucleotide sequence according to the invention is functionally linked with regulatory sequences suitable for expression of the encoded amino acid sequence in a suitable host cell, such as A. niger or A. oryzea . The invention also provides methods for preparing polynucleotides and vectors according to the invention. The invention also relates to recombinantly produced host cells that contain heterologous or homologous polynucleotides according to the invention. In one embodiment, the invention provides recombinant host cells wherein the expression of a protease according to the invention is significantly reduced or wherein the activity of the protease is reduced or wherein the protease is even inactivated. Such recombinants are especially useful for the expression of homologous or heterologous proteins. In another embodiment, the invention provides recombinant host cells wherein the expression of a protease according to the invention is significantly increased or wherein the activity of the protease is increased. Such recombinants are especially useful for the expression of homologous or heterologous proteins where maturation is seriously hampered in case the required proteolytic cleavage becomes the rate limiting step. In another embodiment the invention provides for a recombinantly produced host cell that contains heterologous or homologous DNA according to the invention, preferably DNA encoding proteins bearing signal sequnences and wherein the cell is capable of producing a functional protease according to the invention, preferably a cell capable of over-expressing the protease according to the invention, for example an Aspergillus strain comprising an increased copy number of a gene or cDNA according to the invention. In another embodiment the invention provides for a recombinantly produced host cell that contains heterologous or homologous DNA according to the invention and wherein the cell is capable of secreting a functional protease according to the invention, preferably a cell capable of over-expressing and secreting the protease according to the invention, for example an Aspergillus strain comprising an increased copy number of a gene or cDNA according to the invention. In yet another aspect of the invention, a purified polypeptide is provided. The polypeptides according to the invention include the polypeptides encoded by the polynucleotides according to the invention. Especially preferred is a polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or functional equivalents thereof. The invention also provides for antibodies reactive with a polypeptide according to the invention. These antibodies may be polyclonal, yet especially preferred are monoclonal antibodies. Such antibodies are particularly useful for purifying the polypeptides according to the invention. Fusion proteins comprising a polypeptide according to the invention are also within the scope of the invention. The invention also provides methods of making the polypeptides according to the invention. The invention further relates to a method for diagnosing aspergillosis either by detecting the presence of a polypeptide according to the invention or functional equivalents thereof, or by detecting the presence of a DNA according to the invention or fragments or functional equivalents thereof. The invention also relates to the use of the protease according to the invention in an industrial process as described herein detailed-description description="Detailed Description" end="lead"? |
Compound |
This invention relates to new polypeptide compound represented by the following general formula (I): wherein R1, R2, R3, R4, R5 and R6 are as defined in the description or a salt thereof which has antimicrobial activities (especially, antifungal activities), inhibitory activity on β-1,3-glucan synthase, to process for preparation thereof, to a pharmaceutical composition comprising the same, and to a method for prophylactic and/or therapeutic treatment of infectious diseases including Pneumocystis carinii infection (e.g. Pneumocystis carinii pneumonia) in a human being or an animal. |
1. A polypeptide compound of the following general formula (I): wherein R1 is acyl group, R2 is hydrogen or acyl group, R3 is lower alkyl which has one or more hydroxy or protected hydroxy, R4 is hydrogen or hydroxy, R5 is hydrogen, hydroxy, lower alkoxy or hydroxy sulfonyloxy, and R6 is hydroxy or acyloxy, or a salt thereof: 2. A compound of claim 1, wherein R1 is phenyl(lower)alkenoyl substituted with one or more suitable substituent(s), benzoyl substituted with one or more suitable substituent(s) or naphthoyl substituted with one or more suitable substituent(s), R2 is hydrogen, R3 is lower alkyl which has one or more hydroxy, R4 is hydrogen or hydroxy, R5 is hydroxy or hydroxysulfonyloxy and R6 is hydroxy. 3. A compound of claim 2, wherein R1 is phenyl(lower)alkenoyl substituted with one or more suitable substituent(s), benzoyl substituted with one ore more suitable substituent(s) or naphthoyl substituted with one or more suitable substituent (s), R2 is hydrogen, R3 is lower alkyl which has two hydroxy, R4 is hydrogen or hydroxy; R5 is hydroxy or hydroxysulfonyloxy; and R6 is hydroxy. 4. A compound of claim 3, wherein R1 is naphthoyl substituted with higher alkoxy, naphthoyl substituted with lower alkoxy(higher)alkoxy, naphthoyl substituted with higher alkyl, phenyl(lower)alkenoyl substituted with lower alkoxy, benzoyl substituted with a suitable substituent selected from the group consisting of phenyl substituted with a suitable substituent selected from the group consisting of lower alkoxy, higher alkoxy and higher alkyl, thiadiazolyl substituted with phenyl which has a suitable substituent selected from the group consisting of piperazinyl substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, piperazinyl substituted with lower alkoxy(higher)alkyl, piperazinyl substituted with tetrahydropyran, piperazinyl substituted with dioxaspiro(higher)alkyl which may have lower alkyl, piperazinyl substituted with lower alkyl having pyridyl, piperidyl substituted with lower alkoxy and chlorophenyl, piperidyl substituted with lower alkoxy, piperidyl substituted with lower alkoxy having cyclo(lower)alkyl, piperidyl substituted with lower alkoxy(higher)alkoxy, dioxaazaspiro(higher)alkyl, tetrahydropyrazolopyridyl substituted with phenyl, cyclo(lower)alkyloxy, piperidyloxy substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, piperidyloxy substituted with lower alkoxy(higher)alkyl, piperidyloxy substituted with phenyl which may have lower alkoxy, piperidyl substituted with lower alkoxy higher alkyl, and piperidyl substituted with lower alkoxy(lower)alkoxy, thiadiazolyl substituted with pyridyl having piperidyl substituted with phenyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy(lower)alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy and cyclo(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with phenyl which may have lower alkoxy, imidazothiadiazolyl substituted with phenyl having piperidyloxy substituted with cyclo(lower)alkyl which may have lower alkoxy(lower)alkoxy, imidazothiadiazolyl substituted with phenyl having tetrahydropyridyl substituted with cyclo(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperidyl substituted with lower alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having piperazinyl substituted with lower alkoxy(lower)alkyl, imidazothiadiazolyl substituted with phenyl having lower alkoxy(higher)alkyl, imidazothiazolyl substituted with phenyl having lower alkoxy(lower)alkoxy, phenyl substituted with piperazinyl having phenyl substituted with lower alkoxy, phenyl substituted with piperazinyl having phenyl substituted with piperidyloxy having lower alkoxy(lower)alkyl, phenyl substituted with diazabicyclo(higher)alkyl having cyclo(lower)alkyl, phenyl substituted with hexahydrodiazepinyl having cyclo(lower)alkyl, phenyl substituted with piperidyl having phenyl, phenyl substituted with piperazinyl having phenyl substituted with piperazinyl having lower alkoxy(lower)alkyl, piperazinyl substituted with thiadiazolyl having phenyl substituted with lower alkoxy(higher)alkoxy, thiazolyl substituted with phenyl having lower alkoxy, oxadiazolyl substituted with phenyl having higher alkoxy, oxadiazolyl substituted with phenyl having phenyl substituted with lower alkoxy, oxadiazolyl substituted with phenyl having piperazinyl substituted with cyclo(lower)alkyl having lower alkyl, pyrazolyl substituted with phenyl having phenyl, and pyrazolyl substituted with phenyl having lower alkoxy, R2 is hydrogen, R3 is lower alkyl which has two hydroxy, R4 is hydrogen or hydroxy; R5 is hydroxy or hydroxysulfonyloxy; and R6 is hydroxy. 5. A process for preparing a polypeptide compound (I) of claim 1, or a salt thereof, which comprises, 1) reacting a compound (II) of the formula: wherein R1, R4, R5 and R6 are defined in claim 1, or its reactive derivative at the amino group or a salt thereof, with a compound (III) of the formula: R3═O (III) wherein R3 is defined in claim 1, or its reactive derivative or a salt thereof, to give a compound (Ia) of the formula: wherein R1, R3, R4, R5 and R6 are defined above, or a salt thereof, or ii) reacting a compound (Ia) of the formula: wherein R1, R3, R4, R5 and R6 are defined in claim 1, or its reactive derivative at the amino group or a salt thereof, with a compound (IV) of the formula: Ra2—OH (IV) wherein R1, Ra2 is acyl group, or its reactive derivative at the carboxy group or a salt thereof, to give a compound (Ib) of the formula: wherein R1, Ra2, R3, R4, R5 and R6 are defined above, or a salt thereof, or iii) subjecting a compound (Ib) of the formula: wherein R1, R3, R4, R5 and R6 are defined in claim 1, Ra2 is acyl group, or a salt thereof, to elimination reaction of the acyl group, to give a compound (Ia) of the formula: wherein R1, R3, R4, R5 and R6 are defined above, or a salt thereof, or iv) reacting a compound (Ic) of the formula: wherein R2, R3, R4, R5 and R6 are defined in claim 1, or its reactive derivative at the amino group or a salt thereof, with a compound (V) of the formula: Ra1—OH (V) wherein Ra1 is acyl group, or its reactive derivative at the carboxy group or a salt thereof, to give a compound (Id) of the formula: wherein R2, R3, R4, R5 and R6 are defined in claim 1, Ra1 is defined above, or a salt thereof. 6. A pharmaceutical composition which comprises, as an active ingredient, a compound of claim 1 or a pharmaceutically acceptable salt thereof in admixture with pharmaceutically acceptable carriers or excipients. 7. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament. 8. A compound of claim 1 or a pharmaceutically acceptable salt thereof for use as a medicament. 9. A method for the prophylactic and/or therapeutic treatment of infectious diseases caused by pathogenic microorganisms, which comprises administering a compound of claim 1 or a pharmaceutically acceptable salt thereof to a human being or an animal. 10. A commercial package comprising the pharmaceutical composition of claim 7 and a written matter associated therewith, wherein the written matter states that the pharmaceutical composition can or should be used for preventing or treating infections disease. 11. An article of manufacture, comprising packaging material and the compound (I) identified in claim 1 contained within said packaging material, wherein said the compound (I) is therapeutically effective for preventing or treating infectious diseases, and wherein said packaging material comprises a label or a written material which indicates that said compound (I) can or should be used for preventing or treating infectious diseases. |
<SOH> BACKGROUND ART <EOH>In U.S. Pat. Nos. 5,376,634, 5,569,646, WO 96/11210 and WO 99/40108, there are disclosed the polypeptide compound and a pharmaceutically acceptable salt thereof, which have antimicrobial activities (especially antifungal activity). |
Variable light wave function circuit and variable light wave function device |
Characteristics are rendered variable and high-functional by using the side-pressure inductive polarization mode coupling of a PMF to thereby change the position and magnitude of a side pressure. An input light is incident via a polarizer (2), and an outgoing light is output via the PMF (1) and another polarizer (3). Light may enter and go out in an opposite way. The PMF (1) has two polarization axes orthogonal to each other, and the polarization axis of the polarizer (2) is coupled so as to agree with one end of the polarization axis of the PMF (1). The polarization axis of the polarizer (3) is coupled so as to agree with one end of the polarization axis of the PMF (1). The PMF (1) induces polarization mode coupling when a polarization light tilted a specified angle with respect to the polarization axis is incident to apply a side pressure to the PMF (1). Characterstics/functions can be changed by changing the position and the magnitude of a side pressure by an application unit (5) so that the length of the PMF (1) of a basic structure can be easily set precisely. |
1. A variable lightwave functional circuit comprising: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; a first polarizer arranged at one end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said first polarizer is inclined at a predetermined angle with respect to the polarization axis of said polarization-maintaining optical fiber; and a second polarizer arranged at the other end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said second polarizer is made coincident with the polarization axis of said polarization-maintaining optical fiber; wherein: laser light is entered via any one of said first polarizer and said second polarizer to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of said first polarizer and said second polarizer, which is not arranged on the side of laser entering port thereof. 2. A variable lightwave functional circuit comprising: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; and a branching/coupling device having a first port to a fourth port, for branching laser light inputted from the first port to both said second port and said third port, and also for projecting laser light inputted from said second port to the fourth port; wherein: one end of said polarization-maintaining optical fiber is connected to one of said second port and said third port in such a manner that the polarization axis is inclined at a predetermined angle to an axial direction parallel to a branching plane of said branching/coupling device; the other end of said polarization-maintaining optical fiber is connected to the other of said second port and said third port in such a manner that the polarization axis is made coincident with said axial direction parallel to said branching plane of said branching/coupling device; laser light is entered from the first port of said branching/coupling device via the third port to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from said fourth port of said branching/coupling device. 3. A variable lightwave functional circuit as claimed in claim 2 wherein: said branching/coupling device is a polarization beam splitter and said variable lightwave functional circuit further comprising: a polarizer arranged between either said second port or said third port of said branching/coupling device and said polarization-maintaining optical fiber, the polarization axis of which is adjusted in such a manner that the laser light whose characteristic has been changed passes through said branching/coupling device. 4. A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, further comprising: a rotator for inclining a polarization axis of entered laser light at a predetermined angle with respect to the polarization axis of said polarization-maintaining optical fiber. 5. A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: said applying unit applies the lateral pressure to said polarization-maintaining optical fiber at either one point or plural points in equal intervals. 6. A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: said applying unit applies the lateral pressure to said polarization-maintaining optical fiber at positions defined from a zero-th position to an N-th (symbol “N” being integer) position, where an interval from one end of said polarization-maintaining optical fiber up to the zero-th position is defined as 2NL (symbol “L” being predetermined length), an interval between the zero-th position and a first position is defined as 2N-1L, - - - , an interval between a(k-1)-th position and a k-th position is defined as 2N-kL, - - - , and an interval between an (N-1)-th position and the N-th position is defined as 20L; and said applying unit applies the lateral pressure in such a manner that equal rotations are applied at the respective positions, so that a repetition frequency of a pulse stream of entered laser light is multiplexed. 7. A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: either the characteristic to be changed or a function to be changed includes one of a time period, a center wavelength, a bandwidth; a wavelength to be derived, a repetition frequency, a gain, a group velocity, and a dispersion. 8. A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: the entered laser light corresponds to wavelength division multiplexed laser light. 9. A variable lightwave functional circuit as claimed in any one of claim 1 to claim 3, wherein: a predetermined angle of mutual polarization axes defined between said polarization-maintaining optical fiber and said first polarizer, said second polarizer, or said branching/coupling device is either 45 degrees or approximately 45 degrees. 10. A variable lightwave functional apparatus comprising: an optical amplifier for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a polarization branching/coupling device having a first port to a third port, for branching the output from said optical amplifier, which is entered from said first port into the polarization branching/coupling device, to both said second port and said third port; a coupler arranged at the second port of said polarization branching/coupling device; and the variable lightwave functional circuit recited in any one of claim 1 to claim 3, and arranged between said third port of said polarization branching/coupling device and said coupler; wherein: the wavelength multiplexed light containing said plural pulses having said first wavelength interval is converted into wavelength multiplexed light containing a plurality of pulses having a second wavelength interval in response to lateral pressure applied to said polarization-maintaining optical fiber, and then said converted wavelength multiplexed light is outputted from said coupler 11. A variable lightwave functional apparatus comprising: an acousto-optical modulator for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a coupler arranged at an output of said acousto-optical modulator; a polarization branching/coupling device having a first port to a third port, for branching the output form said coupler, which is entered from said first port into the polarization branching/coupling device, to both said second port and said third port; an optical amplifier connected to said second port of said polarization branching/coupling device; and the variable lightwave functional circuit recited in any one of claim 1 to claim 3, and arranged between said second port of said polarization branching/coupling device and said optical amplifier; wherein: multi-wavelength oscillation light is outputted from said coupler by laser light from said acousto-optical modulator. 12. A variable lightwave functional apparatus as claimed in claim 10, further comprising: a polarizer arranged between either said second port or said third port of said polarization branching/coupling device and said polarization-maintaining optical fiber, for adjusting a polarization axis thereof in such a manner that laser light whose characteristic has been changed is transmitted by said polarization branching/coupling device. 13. A variable lightwave functional apparatus as claimed in claim 11, further comprising: a polarizer arranged between either said second port or said third port of said polarization branching/coupling device and said polarization-maintaining optical fiber, for adjusting a polarization axis thereof in such a manner that laser light whose characteristic has been changed is transmitted by said polarization branching/coupling device. 14. A method of making a variable lightwave functional circuit comprising: providing an applying unit for applying lateral pressure; providing a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; providing a first polarizer arranged at one end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said first polarizer is inclined at a predetermined angle with respect to the polarization axis of said polarization-maintaining optical fiber; and providing a second polarizer arranged at the other end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said second polarizer is made coincident with the polarization axis of said polarization-maintaining optical fiber; wherein laser light is entered via any one of said first polarizer and said second polarizer to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of said first polarizer and said second polarizer, which is not arranged on the side of laser entering port thereof 15. A method for making a variable lightwave functional circuit, said method comprising: providing an applying unit for applying lateral pressure; providing a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; providing a branching/coupling device having a first port to a fourth port, for branching laser light inputted from the first port to both said second port and said third port, and also for projecting laser light inputted from said second port to the fourth port; wherein one end of said polarization-maintaining optical fiber is connected to one of said second port and said third port in such a manner that the polarization axis is inclined at a predetermined angle to an axial direction parallel to a branching plane of said branching/coupling device; the other end of said polarization-maintaining optical fiber is connected to the other of said second port and said third port in such a manner that the polarization axis is made coincident with said axial direction parallel to said branching plane of said branching/coupling device; and laser light is entered from the first port of said branching/coupling device via the third port to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from said fourth port of said branching/coupling device. 16. A method of using a variable lightwave functional circuit, said variable lightwave functional circuit comprising: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; a first polarizer arranged at one end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said first polarizer is inclined at a predetermined angle with respect to the polarization axis of said polarization-maintaining optical fiber; and a second polarizer arranged at the other end of said polarization-maintaining optical fiber in such a manner that a polarization axis of said second polarizer is made coincident with the polarization axis of said polarization-maintaining optical fiber; wherein laser light is entered via any one of said first polarizer and said second polarizer to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of said first polarizer and said second polarizer, which is not arranged on the side of laser entering port thereof; said method comprising transmitting laser light into said polarization-matching optical fiber; and applying lateral pressure to said polarization-matching optical fiber. 17. A method of using a variable lightwave functional circuit, said variable lightwave functional circuit comprising: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by said applying unit; a branching/coupling device having a first port to a fourth port, for branching laser light inputted from the first port to both said second port and said third port, and also for projecting laser light inputted from said second port to the fourth port; wherein one end of said polarization-maintaining optical fiber is connected to one of said second port and said third port in such a manner that the polarization axis is inclined at a predetermined angle to an axial direction parallel to a branching plane of said branching/coupling device; the other end of said polarization-maintaining optical fiber is connected to the other of said second port and said third port in such a manner that the polarization axis is made coincident with said axial direction parallel to said branching plane of said branching/coupling device; laser light is entered from the first port of said branching/coupling device via the third port to said polarization-maintaining optical fiber; said polarization-maintaining optical fiber changes a characteristic related to either a transmission wavelength or a repetition frequency of a pulse stream by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by said applying unit, or plural conditions thereof; and said polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from said fourth port of said branching/coupling device; said method comprising transmitting laser light into said polarization-matching optical fiber; and applying lateral pressure to said polarization-matching optical fiber. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention is related to a variable lightwave functional circuit and a variable lightwave functional apparatus. More specifically, the present invention is directed to a variable lightwave functional circuit and a variable lightwave functional apparatus, using a mode coupling within a polarization-maintaining optical fiber. Network technology of optical fiber communication systems has been conducted in practical fields in connection with appearances of optical fiber amplifiers in a front half decade in 1990, and is being rapidly developed. In the present stage, there are certain possibilities that ultra-long distance (e.g., up to 10,000 Km) and very high-speed (e.g., up to 40 Gb/s) wavelength division multiplexed transmission systems (WDM system operable up to, e.g., 64 wavelengths) could be realized. Then, optical fiber information communication networks may be conceivable as the most important infrastructure in the beginning stage of 21 century. However, up to now, while optical fiber communication systems are mainly employed only in point-to-point correspondence trunk line systems, conventional coaxial cables and semiconductor integrated circuits/electronic devices may constitute major basic elements in network portions. Since the Internet has been currently developed in explosive manners, extensions of transfer capacities which are required for future's networks could be predicted by that the transfer capacities are extended twice per 12 months. This extension rate of the transfer capacities exceeds a so-called “Moore's law (twice per 18 months)” related to semiconductor integrated circuits. Soon or later, there is no doubt about such a fact that WDM optical networks using light are necessarily required also in these network portions. However, under the present situation, optical devices employed in such WDM optical networks have not yet been well-developed, so that very rapid development of these optical devices is necessarily needed. |
<SOH> SUMMARY OF THE INVENTION <EOH>That is to say, more specifically, the present invention is featured by realizing a “variable optical transversal filter using a lateral-pressure induced polarization mode of a polarization-maintaining optical fiber (PMF)”. In general, a transversal filter is equipped with a repeated structure and a summation circuit, in which a delay line and a branching/weighting circuit are employed as a basic unit. Different from an electric summation circuit, since a summation circuit can be hardly realized in an optical field, a two-port-pair cascade connecting mode optical lattice type filter may be employed [1] (note that symbol “[ ]” indicates below-mentioned reference publication number). Also, the optical lattice filter constructed of the planar lightwave circuit (PLC) with employment of the repeated Mach-Zehnder optical circuit structure has been proposed by JINGUJI et al. of NTT [2], and various sorts of the above-explained functions have be realized. Also, such a fact has been disclosed by KOSEKI of Sophia University. That is, the optical lattice filter could be manufactured even by the PMF rotational connection structure which utilizes the delays occurred between the polarization modes of the PMF as the delay, and also the rotational connection of the PMF as the branching/weighting operations [1] and [3]. However, as to these structures which have been conventionally realized, the functions and the characteristics of these structures have already been determined when the structures are manufactured, so that these functions and characteristics cannot be changed. Also, in the PMF rotational connection structures, it is practically difficult that the PMF lengths of the basic structures cannot be made coincident in higher precision. The present invention has been made to solve the above-described difficulties, and has an object to provide both an optical fiber type variable lightwave functional circuit and a variable lightwave functional apparatus, capable of properly accepting structural changes in a WDM optical network, and furthermore, capable of having flexibilities as to various functions. As a consequence, the present invention owns such an object to provide both a variable lightwave functional circuit and a variable lightwave functional apparatus, which are capable of realizing a large number of functions, and also can be applied to optical communication apparatus such as WDM optical networks and optical transmitting/receiving devices. These various functions involve optical filtering, wavelength add/drop operation, pulse multiplexing operation, optical amplifier gain equalizing operation, optical fiber wavelength dispersion compensating operation, and the like. Also, an object of the present invention is to provide both a variable lightwave functional circuit and a variable lightwave functional apparatus, such as an optical transversal filter whose characteristic is variable and which owns higher functions, while a lateral-pressure induced polarization mode coupling of a PMF is used instead of a PMF rotational connection, and thus, both a position and a magnitude of lateral pressure are changed. According to a first solving means of the present invention, such a variable lighwave functional circuit is provided which is comprised of: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by the applying unit; a first polarizer arranged at one end of the polarization-maintaining optical fiber in such a manner that a polarization axis of the first polarizer is inclined at a predetermined angle with respect to the polarization axis of the polarization-maintaining optical fiber; and a second polarizer arranged at the other end of the polarization-maintaining optical fiber in such a manner that a polarization axis of the second polarizer is made coincident with the polarization axis of the polarization-maintaining optical fiber; in which: laser light is entered via any one of the first polarizer and the second polarizer to the polarization-maintaining optical fiber, the polarization-maintaining optical fiber changes a characteristic of the variable lighwave functional circuit by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by the applying unit, or plural conditions thereof; and the polarization-maintaining optical fiber projects the laser light whose characteristic has been changed through any one of the first polarizer and the second polarizer, which is not arranged on the side of laser entering port thereof. According to a second solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an applying unit for applying lateral pressure; a polarization-maintaining optical fiber having a polarization axis, for inducing a polarization mode coupling by applying the lateral pressure to a predetermined position by the applying unit; and a branching/coupling device having a first port to a fourth port, in which laser light entered from the first port is branched to both the second port and the third port; one end of the polarization-maintaining optical fiber is arranged between the second port and the third port in such a manner that polarization axes thereof are inclined at a predetermined angle to the branching/coupling device; the other end of the polarization-maintaining optical fiber is arranged in such a manner that polarization axes thereof are made coincident with the branching/coupling device; and laser light entered from either the first port or the second port is projected to the fourth port; in which: laser light is entered from the third port of the branching/coupling device into the polarization-maintaining optical fiber; the polarization-maintaining optical fiber changes a characteristic of the variable lightwave functional circuit by the induced polarization mode coupling in response to either a single condition of a position, a quantity, and a magnitude of lateral pressure applied by the applying unit, or plural conditions thereof; and the polarization-maintaining optical fiber projects the laser light whose characteristic has been changed from the fourth port of the branching/coupling device. According to a third solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an optical amplifier for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a polarization branching/coupling device having a first port to a third port, for branching the output from the optical amplifier, which is entered from the first port into the polarization branching/coupling device, to both the second port and the third port; a coupler arranged at the second port of the polarization branching/coupling device; and the above-described variable lightwave functional circuit arranged between the third port of the polarization branching/coupling device and the coupler; in which: the wavelength multiplexed light containing the plural pulses having the first wavelength interval is converted into wavelength multiplexed light containing a plurality of pulses having a second wavelength interval in response to lateral pressure applied to the polarization-maintaining optical fiber, and then the converted wavelength multiplexed light is outputted from the coupler. According to a fourth solving means of the present invention, such a variable lightwave functional apparatus is provided which is comprised of: an acousto-optical modulator for outputting wavelength multiplexed light containing a plurality of pulses having a first wavelength interval; a coupler arranged at an output of the acousto-optical modulator; a polarization branching/coupling device having a first port to a third port, for branching the output form the coupler, which is entered from the first port into the polarization branching/coupling device, to both the second port and the third port; an optical amplifier connected to the second port of the polarization branching/coupling device; and the above-described variable lightwave functional circuit arranged between the second port of the polarization branching/coupling device and the optical amplifier; in which: multi-wavelength oscillation light is outputted from the coupler by laser light from the acousto-optical modulator. |
Use of n-acetyl-d-glucosamine in the manufacture of pharmaceutical useful for adjuvant treatment of perianal disease |
The present invention has disclosed a use of N-acetyl-D-glucosamine in the manufacture of a medicine for auxiliary treatment of the peri-anal disease. Through stabilizing celluar lysosome membrance, the degree and scope of the injure extended by the release of various enzymes in the cellular lysosome are decreased; improving the healing of the injured tissues; resist the field planting of the microorganism on the traumatic surface so as to prevent the occurrence of infection. The preparation with N-acetyl-D-glucosamine as a main active component can be used in the auxiliary treatment of the peri-anal diseases, with a remarkable curative effect. |
1-4. (canceled) 5. A method for treating perianal tissue injury, comprising: administering to the site of the perianal tissue injury a therapeutically effective amount of a medicament that contains N-acetyl-D-glucosamine and/or a pharmaceutically acceptable salt thereof. 6. A method as recited in claim 5, wherein the medicament is in the form of a liquid. 7. A method as recited in claim 5, wherein the medicament is sprayed on the injured tissue. 8. A method as recited in claim 5, wherein the medicament is applied to a subject's perineum. 9. A method as recited in claim 5, wherein the medicament is applied to a subject's perianal mucousal membrane. |
<SOH> BACKGROUND ART <EOH>The peri-anal disease includes anal fissure, peri-anal abscess, fistula cannulas, haemorrhoids, polyp of rectum, carcinoma of the rectum and so on, the common feature of which is that there is an tissue injure in-situ, so, normally, the symptom is light, but when it acutely break out, there will be pain, red and swollen, pruritus, exudation increasing and so on, which would bring many worries and pains to the people's life and work. The treatment of these diseases needs eliminating acute oedema, lightening pain, stabilizing membrane structure, so as to prevent the inflammation to be more intensive, at the same time, it is needed to control the infection. Said disease is known as a little disease but a big problem clinically for many years. Therefore, the medicament for auxiliary treatment of peri-anal disease is needed all along in the field. In the research of “bio-waves” theory, the present inventor has set up a bacterial wave growth model. Through researching, it is known that this wave is of its intrinsic regulation mechanism: some chemical substances are able to participate the regulation in the bio-wave process, so as to transform an abnormal periodic slow wave into a normal physiological chaotic quick wave, and this kind of substances are known as promoting wave factors. Through separating, purifying and identifying, it is determined that one of the factors is N-acetyl-D-glucosamine, the promoting wave function of which is shown in lubricating and protecting the cell. Many biochemical and physiological process of human body need the participation of the promoting wave factors, and it would lead to an abnormal state, if this kind of promoting wave factors is lacked in the living body. N-acetyl-D-glucosamine is a chemical reagent. From the 1990's, it is continually used to treat pericementitis (WO9102530A1), microbiological infection (WO9718790A3), intestinal inflammation (WO9953929A1), cornea disease (JP10287570A2), hypertrophy of the prostate (U.S. Pat. No. 5,116,615) and so on. It is also applied in cosmetology (JP59013708A2), shampoo preparation (JP2011505A2), tissue growth regulation agent (WO/A 8 702244), and etc., but it has not been used in the manufacture of a medicament for auxiliary treatment of peri-anal disease up to now. |
Apparatus for heating a food product and heating devices and a feed assembly therefor |
A device (22) for heating a food product (P). The device (22) including an oven enclosure (52) at lest one magnetron for emitting microwave energy and a microwave energy focusing device (68, 70, 72) associated with at least one magnetron (56, 58, 60, 62, 64 and 66) and adapted to focus microwave energy towards the food product. The magnetron(s) is/are disposed external the enclosure (52) and the microwave energy focusing device(s) is/are disposed internal the enclosure (52). Also disclosed is a food product support device (116) of a substantially truncated conical external shape with a first larger base surface (118) adapted to be positioned adjacent the base of the oven enclosure and a second smaller supporting surface (120) adapted to support a food product (P) in a position vertically displaced from the base of the oven enclosure for heating and a sloping side surface (122) extending between the base and support surfaces. The food product (P) is able to exit the oven enclosure by sliding down the side surface (122) when pushed from the support surface towards (120) an opening in the oven enclosure. Also disclosed is a food product positioning and ejection device (138) adapted for reciprocal movement towards and away from an opening in the oven enclosure. |
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