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Dock 3 tumor suppressor gene |
The invention relates to a newly identified tumor suppressor gene, designated DOS (for Deleted in Osteosarcoma and alternatively referred to herein as DOCK 3) which has been cloned from human and mouse cells. The DOS nucleic acid and protein molecules and their use in the diagnosing and treating disorders characterized by aberrant DOS molecule expression are described. |
1. An isolated nucleic acid molecule selected from the group consisting of: (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO:1 or SEQ ID NO:3, and which code for a DOS protein, (b) deletions, additions and substitutions of the nucleic acid molecules of (a), (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c). 2. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule comprises SEQ ID NO:1. 3. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule comprises SEQ ID NO:3. 4. An isolated nucleic acid molecule selected from the group consisting of: (a) a unique fragment of the nucleotide sequence set forth as SEQ ID NO:1 or set forth as SEQ ID NO:3 between 12 and 115 nucleotides in length or more, and (b) complements of (a), wherein the unique fragments exclude nucleic acids having nucleotide sequences that are contained within SEQ ID NO:1 or SEQ ID NO:3, and that are known as of the filing date of this application. 5. The isolated nucleic acid molecule of claim 4 wherein the isolated nucleic acid molecule comprises SEQ ID NO: 31. 6. An isolated nucleic acid molecule selected from the group consisting of: (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29, (b) deletions, additions and substitutions of the nucleic acid molecules of (a), (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c). 7. An expression vector comprising the isolated nucleic acid molecule of claim 1 operably linked to a promoter. 8. A host cell transformed or transfected with the expression vector of claim 7. 9. A transgenic non-human animal comprising the expression vector of claim 7. 10. A transgenic non-human animal which has reduced expression of a DOS nucleic acid molecule or of a Mutant DOS nucleic acid molecule. 11. An isolated protein encoded by the isolated nucleic acid molecule of claim 1. 12. The isolated protein of claim 11, wherein the isolated protein comprises of the amino acid sequence of selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. 13. The isolated protein of claim 11 wherein the isolated protein comprises SEQ ID NO: 32. 14. A binding polypeptide that selectively binds to the isolated protein of claim 11. 15-17. (canceled) 18. A composition comprising: the nucleic acid of claim 1, and a pharmaceutically acceptable carrier. 19. A composition comprising: the protein encoded by the isolated nucleic acid molecule of claim 1, and a pharmaceutically acceptable carrier. 20. A composition comprising: the binding polypeptide of claim 14, and a pharmaceutically acceptable carrier. 21. A method for making a medicament, comprising: placing an active agent selected from the group consisting of: (a) the isolated nucleic acid molecules of claim 1, (b) the isolated protein of claim 11, and (c) the binding polypeptides of claim 14, in a pharmaceutically acceptable carrier. 22. The method of claim 21, wherein placing comprises placing a therapeutically effective amount of the active agent in the pharmaceutically acceptable carrier to form one or more doses. 23. A method for diagnosing a disorder characterized by aberrant expression of a DOS molecule, comprising: detecting in a first biological sample obtained from a subject, expression of a DOS molecule or a Mutant DOS molecule, wherein decreased expression of a DOS molecule or the increased expression of a Mutant DOS molecule compared to a control sample indicates that the subject has a disorder characterized by aberrant expression of a DOS molecule. 24-25. (canceled) 26. The method of claim 23, wherein the disorder characterized by aberrant expression of a DOS molecule is selected from the group consisting of: a cancer, a tumor, a cytoskeleton disorder, and a cell migration disorder. 27-43. (canceled) 44. A kit for diagnosing a disorder associated with aberrant expression of a DOS molecule, comprising: one or more nucleic acid molecules that hybridize to a DOS nucleic acid molecule or to a Mutant DOS nucleic acid molecule under stringent conditions, one or more control agents, and instructions for the use of the nucleic acid molecules, and agents in the diagnosis of a disorder associated with aberrant expression of a DOS molecule. 45-46. (canceled) 47. A kit for diagnosing a DOS tumor in a subject comprising: one or more binding polypeptides that selectively bind to a DOS protein or a Mutant DOS protein, one or more control agents, and instructions for the use of the binding polypeptides, and agents in the diagnosis of a disorder associated with aberrant expression of a DOS molecule. 48-50. (canceled) 51. A method for treating a subject with a disorder characterized by aberrant expression of a DOS molecule, comprising administering to the subject an effective amount of a DOS nucleic acid molecule to treat the disorder. 52. A method for treating a subject with a disorder characterized by aberrant expression of a DOS molecule, comprising administering to the subject an effective amount of an anti-sense molecule to a Mutant DOS nucleic acid molecule to treat the disorder. 53. (canceled) 54. A method for treating a subject with a disorder characterized by aberrant expression of a DOS molecule, comprising administering to the subject an effective amount of a DOS protein to treat the disorder. 55. A method for treating a subject with a disorder characterized by aberrant expression of a DOS molecule, comprising administering to the subject an effective amount of a binding polypeptide to a Mutant DOS protein to treat the disorder. 56-58. (canceled) 59. A method for producing a DOS protein comprising providing a DOS nucleic acid molecule operably linked to a promoter, wherein the DOS nucleic acid molecule encodes the DOS protein or a fragment thereof, expressing the DOS nucleic acid molecule in an expression system, and isolating the DOS protein or a fragment thereof from the expression system. 60. (canceled) 61. A method for producing a Mutant DOS protein comprising providing a Mutant DOS nucleic acid molecule operably linked to a promoter, wherein the Mutant DOS nucleic acid molecule encodes the Mutant DOS protein or a fragment thereof, expressing the Mutant DOS nucleic acid molecule in an expression system, and isolating the Mutant DOS protein or a fragment thereof from the expression system. 62. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Cancer progression is caused by accumulation of multiple mutations that provide selective advantage during cancer growth, invasion and metastasis (1, 2, 3). While gain of function mutations occur in oncogenes, many of the genetic events that underlie cancer appear to be inactivating, or loss of function mutations affecting tumor suppressor genes (1). Tumor suppressor gene identified to date exhibit diverse cellular functions (4). Functional studies on these tumor suppressor genes have supported the original hypothesis that these genes represent potential bottlenecks in wide variety of cellular pathways (4). These include proliferation, differentiation, apoptosis and response to DNA damage. For example p53 and WT1 are DNA binding transcription factors; RB1, APC and possibly BRCA1 indirectly modulate transcription; P16 is an inhibitor of kinases required for cell cycle progression; PTEN is a novel phosphatase; NF2 is a cell structural component; VHL is a potential mediator of mRNA processing. A large number of genes are believed to be genomic caretakers and mutations in these genes cause microsatellite instability (MSH2, MLH1, PMS1 and PMS2) or chromosomal instability (p53, possibly BRCA1 and BRCA2). To date, genes involved in advanced stages of cancer progression such as invasion, angiogenesis and metastasis have not been identified (4, 5). They are likely to become evident over time with large-scale genome wide analysis. The vast majority of cancers result from sporadic genetic events and only rare cases (less than 1%) have an inherited component (7, 8). However, the isolation of tumor suppressor genes has typically originated from genetic analysis of such rare inherited cancer syndromes (7). Linkage analysis on large families with cancer present in multiple generations allows identification of markers that co-segregate with cancer. In some cases cytogenetic abnormalities could also be observed either in sporadic or in germline tumors (7,9). For example, a small fraction of retinoblastomas have a homozygous deletion of RB1 gene (7). Rare Wilms tumor and colon cancer have deletions of WT1 and APC, respectively. These germline or sporadic homozygous deletions have been instrumental in tumor suppressor gene cloning efforts (7). Allelic losses in tumors are typically detected as “loss of heterozygosity” or “LOH”. This represents loss of a polymorphic marker, commonly resulting from a large interstitial deletion or chromosomal non-disjunction event. While LOH is a common event in cancer, it only allows rough mapping of tumor suppressor loci (9). The large size of the LOH region (>10 Mb) makes the identification of the specific tumor suppressor gene targeted by mutation difficult. In contrast, homozygous deletions in tumors are typically small (<100 Kb) since they are restricted by the deletion of the flanking genes. Homozygous deletions occur by diverse mechanisms, including a small deletion in one allele accompanied by LOH of the second allele, or even large deletion of each allele whose common region of overlap is small. Identification of such homozygous deletions can be a powerful approach to identify tumor suppressor genes (7). Significant technological advances have been made to identify regions of chromosomes involved in tumor progression. Analyses of metaphase chromosomes show chromosomal rearrangements in leukemia and lymphomas (10). This is more difficult in solid tumors where karyotyping is less commonly performed. Fluorescence in situ hybridization (FISH) has greatly improved the sensitivity and specificity of detecting chromosome aberrations (11, 12). However, its application in human malignancies is still limited because of complex karyotypes seen in clinical samples. Comparative genome hybridization (CGH) uses both normal and tumor genomes to identify regions in tumor DNA that have undergone changes in copy number (13). In this technique, normal and tumor DNA are labeled with two different haptens that fluoresce at different wavelengths. The probes are then hybridized to metaphase chromosomes in the presence of excess Cot-1 DNA thus inhibiting hybridization of labeled repetitive sequences. The ratio of the amount of two genomes that hybridize to specific areas of the chromosomes indicates the copy number of the two samples. CGH is currently limited to a resolution of 10 to 20 Mb and more sensitive in detecting amplifications rather than a small deletion (9). An alternative method, Representational Difference Analysis (RDA) is a PCR based subtractive hybridization technique, that is particularly applicable in isolating homozygous deletions in tumors (14, 18, 19). It has already been successful in isolating tumor suppressor genes PTEN and DMBT1 and has played a significant role in cloning of BRCA2 (15, 16, 17). In view of the foregoing, a need exists to identify novel tumor suppressor genes to detect and treat various cancers. Preferably, such suppressor genes will have unique sequences that will permit the targeting of therapeutic agents for treating such cancers and the development of agents for detecting such cancers. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides novel human and mouse tumor suppressor genes and is based, in part, on the discovery that this novel gene is deleted in a mouse osteosarcoma cell line. Using Representational Difference Analysis (RDA) on a mouse tumor model, we found a region of homozygous deletion in the mouse cell line. This region of deletion is homologous to human chromosome 7q31. We have cloned the gene residing in the deleted segment (DOS, for deleted in osteosarcoma, alternatively referred to herein as DOCK 3) from both mouse and humans. Human and mouse DOS protein sequence is about 97% identical. The protein has limited homology (approximately 30% identity) with three known genes, namely, DOCK180, myoblast city, and Ced 5. These three proteins are evolutionary conserved in both sequence and function and regulate actin cytoskeleton during cell migration. Accordingly, although not wishing to be bound to any particular theory or mechanism, we believe the DOS gene plays a role in regulating actin cytoskeleton in cell growth and cancer. Thus, the invention is directed to novel compositions of the DOS nucleic acids and proteins encoded thereby, as well as to agents that selectively bind to these novel molecules, and to diagnostic, therapeutic, and research applications of these compositions. According to one aspect of the invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of: (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO:1 or SEQ ID NO:3, and which code for a DOS protein, (b) deletions, additions and substitutions of the nucleic acid molecules of (a), (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c). The preferred isolated nucleic acids of the invention are DOS nucleic acid molecules which encode a DOS protein. As used herein, a DOS protein refers to a protein which is encoded by a nucleic acid having SEQ ID NO:1 or SEQ ID NO:3, or a functional fragment thereof, provided that the functional fragment encodes a protein which plays a role in tumor suppression, cytoskeletal organization, cell proliferation, cell migration, cellular growth and development, and/or cell-cell interaction. In the preferred embodiments, the isolated nucleic acid molecule is SEQ ID NO:1 or SEQ ID NO:3 According to another aspect of the invention, further isolated nucleic acid molecules that are based on the above-noted DOS nucleic acid molecules are provided. In this aspect, the isolated nucleic acid molecules are selected from the group consisting of: (a) a unique fragment of the nucleotide sequence set forth as SEQ ID NO:1 or set forth as SEQ ID NO:3 between 12 and 115 nucleotides in length or more and (b) complements of (a), wherein the unique fragments exclude nucleic acids having nucleotide sequences that are contained within SEQ ID NO:1 or SEQ ID NO:3, and that are known as of the filing date of the priority application. In one embodiment of the invention an isolated unique nucleic acid fragment comprising SEQ ID NO: 31 of SEQ ID NO: 1 is provided. In yet another aspect of the invention, Mutant DOS nucleic acid molecules are provided. The Mutant DOS nucleic acid molecules contain a sequence which is identical to SEQ ID NO:1 or SEQ ID NO:3, with the exception that the sequence includes one or more mutations, e.g., point mutations, deletion mutations, such that the Mutant DOS nucleic acid molecule does not encode a functional DOS protein. Rather, the Mutant DOS nucleic acid molecules encode a Mutant DOS protein, i.e., a protein which does not exhibit a DOS protein functional activity. In preferred embodiments, the binding polypeptide is an antibody or antibody fragment, more preferably, an Fab or F(ab) 2 fragment of an antibody. Typically, the fragment includes a CDR3 region that is selective for the DOS protein or Mutant DOS protein. Any of the various types of antibodies can be used for this purpose, including monoclonal antibodies, humanized antibodies and chimeric antibodies. According to a further aspect of the invention, pharmaceutical compositions containing the nucleic acids, proteins, and binding polypeptides of the invention are provided. The pharmaceutical compositions contain any of the foregoing therapeutic agents in a pharmaceutically acceptable carrier. Thus, in a related aspect, the invention provides a method for forming a medicament that involves placing a therapeutically effective amount of the therapeutic agent in the pharmaceutically acceptable carrier to form one or more doses. According to another aspect of the invention, various diagnostic methods are provided. In general, the methods are for diagnosing “a disorder characterized by aberrant expression of a DOS molecule”. As used herein, “aberrant expression” refers to either or both of a decreased expression (including no expression) of the DOS molecule (nucleic acid or protein) or an increased expression of a “Mutant DOS molecule”. A Mutant DOS molecule refers to a DOS nucleic acid molecule which includes a mutation (point mutation, deletion) or to a DOS protein molecule (e.g., gene product of mutant DOS nucleic acid molecule) which includes a mutation, provided that the mutation results in a mutant DOS protein that does not have the DOS functional activity that is exhibited by a DOS protein as described herein. The diagnostic methods of the invention can be used to detect the presence of a disorder associated with aberrant expression of a DOS molecule, as well as to assess the progression and/or regression of the disorder such as in response to treatment (e.g., chemotherapy, radiation). According to this aspect of the invention, the method for diagnosing a disorder characterized by aberrant expression of a DOS molecule involves: detecting in a first biological sample obtained from a subject, expression of a DOS molecule or a Mutant DOS molecule; wherein decreased expression of a DOS molecule or the increased expression of a Mutant DOS molecule compared to a control sample indicates that the subject has a disorder characterized by aberrant expression of a DOS molecule. As used herein, a “disorder characterized by aberrant expression of a DOS molecule” refers to a disorder in which there is a detectable difference in the expression levels of DOS molecule(s) and/or Mutant DOS molecule(s) in selected cells of a subject compared to the According to another aspect of the invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of: (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NOs:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29, (b) deletions, additions and substitutions of the nucleic acid molecules of (a), (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c) According to yet another aspect of the invention, an expression vector comprising any of the isolated nucleic acid molecules of the invention operably linked to a promoter are provided. In a related aspect, host cells transformed or transfected with such expression vectors also are provided. According to still a further aspect of the invention, a transgenic non-human animal comprising an expression vector of the invention is provided. Also provided is a transgenic non-human animal which has reduced expression of a DOS nucleic acid molecule or of a Mutant DOS nucleic acid molecule. According to another aspect of the invention, an isolated polypeptide encoded by any of the foregoing isolated nucleic acid molecules of the invention is provided. Preferably, the isolated polypeptide comprises a polypeptide sequence selected from the group, consisting of SEQ ID NO: 2, 4, 9, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. In one aspect of the invention a functional protein fragment of SEQ ID NO: 2 comprising SEQ ID NO: 32 is provided. In yet a further aspect of the invention, binding polypeptides that selectively bind to a DOS molecule and/or to a Mutant DOS molecule are provided. According to this aspect, the binding polypeptides bind to an isolated nucleic acid or protein of the invention, including binding to unique fragments thereof. Preferably, the binding polypeptides bind to a DOS protein, a Mutant DOS protein, or a unique fragment thereof. In certain particularly preferred embodiments, the binding polypeptide binds to a DOS protein but does not bind to a Mutant DOS protein, i.e., the binding polypeptides are selective for binding to the Mutant protein and can be used in various assays to detect the presence of the Mutant DOS protein without detecting DOS protein. control levels of these molecules. Thus, a disorder characterized by aberrant expression of a DOS molecule embraces underexpression (including no expression) of a DOS nucleic acid molecule or a DOS protein compared to control levels of these molecules, as well as overexpression of a Mutant DOS nucleic acid molecule or Mutant DOS protein compared to control levels of these molecules. Such differences in expression levels can be determined in accordance with the diagnostic methods of the invention as disclosed herein. Exemplary disorders that are characterized by aberrant expression of a DOS molecule include: various cancers and disorders associated with abnormal cytoskeleton organization, cell proliferation, cell migration, cellular growth and development, and/or cell-cell interaction. In certain embodiments, the methods of the invention are to diagnose a cancer including, but not limited to, biliary tract cancer, brain cancer (including glioblastomas and medulloblastomas), breast cancer; cervical cancer; choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms, including acute lymphocytic and myelogenous leukemia, multiple myeloma, AIDS associated leukemias and adult T-cell leukemia lymphoma, intraepithelial neoplasms, including Bowen's disease and Paget's disease, liver cancer, lung cancer, lymphomas, including Hodgkin's disease and lymphocytic lymphomas, neuroblastomas, oral cancer, including squamous cell carcinoma, ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer, renal cancer including adenocarcinoma and Wilms tumor, sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma, skin cancer, including melanoma, Kaposi's sarcoma, basocellular cancer and squamous cell cancer, testicular cancer, including germinal tumors (seminomas, and non-seminomas such as teratomas and choriocarcinomas), stromal tumors and germ cell tumors, and thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma. In the preferred embodiments, the methods of the invention are useful for diagnosing Wilms tumor, ovarian carcinoma, renal cell carcinoma, osteosarcoma fibrosarcoma, prostate cnacer, colon cancer, and brain cancer. In yet other embodiments, the diagnostic methods are useful for diagnosing the progression of a disorder. According to these embodiments, the methods further involve: detecting in a second biological sample obtained from the subject, expression of a DOS molecule or a Mutant DOS molecule, and comparing the expression of the DOS molecule or the Mutant DOS molecule in the first biological sample and the second biological sample. In these embodiments, a decrease in the expression of the DOS molecule in the second biological sample compared to the first biological sample or an increase in the expression of the Mutant DOS molecule in the second biological sample compared to the first biological sample indicates progression of the disorder. In yet other embodiments, the diagnostic methods are useful for diagnosing the regression of a disorder. According to these embodiments, the methods further involve: detecting in a second biological sample obtained from the subject, expression of a DOS molecule or a Mutant DOS molecule, and comparing the expression of the DOS molecule or the Mutant DOS molecule in the first biological sample and the second biological sample. In these embodiments, an increase in the expression of the DOS molecule in the second biological sample compared to the first biological sample or a decrease in the expression of the Mutant DOS molecule in the second biological sample compared to the first biological sample indicates regression of the disorder. In certain embodiments, the diagnostic methods of the invention detect a DOS molecule that is a DOS nucleic acid molecule or a Mutant DOS nucleic acid molecule as described above. In yet other embodiments, the methods involve detecting a DOS protein or Mutant DOS protein as described above. Various detection methods can be used to practice the diagnostic methods of the invention. For example, when the methods can involve contacting the biological sample with an agent that selectively binds to the DOS molecule or to the Mutant DOS molecule to detect these molecules. In certain embodiments, the DOS molecule is a nucleic acid and the method involves using an agent that selectively binds to the DOS molecule or to the Mutant DOS molecule, e.g., a nucleic acid that hybridizes under stringent conditions to a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29. In yet other embodiments, the DOS molecule is a protein and the method involves using an agent that selectively binds to the DOS molecule or to the Mutant DOS molecule, e.g., a binding polypeptide, such as an antibody, that selectively binds to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. In yet another embodiment an agent that selectively binds a fragment of a DOS molecule or to a fragment of a Mutant DOS molecule is provided. In certain embodiments the fragment of the DOS molecule or the fragment of the DOS molecule or the fragment of the Mutant DOS molecule is a nucleic acid molecule. One preferred nucleic acid fragment of the DOS molecule compressers SEQ ID NO: 31. In other embodiments, the fragment of the DOS molecule or the fragment of the Mutant DOS molecule is a polypeptide. One preferred polypeptide of the DOS molecule comprises SEQ ID NO: 32. According to still another aspect of the invention, kits for performing the diagnostic methods of the invention are provided. The kits include nucleic acid-based kits or protein-based kits. According to the former embodiment, the kits include: one or more nucleic acid molecules that hybridize to a DOS nucleic acid molecule or to a Mutant DOS nucleic acid molecule under stringent conditions; one or more control agents; and instructions for the use of the nucleic acid molecules, and agents in the diagnosis of a DOS tumor. As used herein, A DOS tumor is disorder associated with aberrant expression of a DOS molecule. Nucleic acid-based kits optionally further include a first primer and a second primer, wherein the first primer and the second primer are constructed and arranged to selectively amplify at least a portion of an isolated DOS nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29. Alternatively, protein based-kits are provided. Such kits include: one or more binding polypeptides that selectively bind to a DOS protein or a Mutant DOS protein; one or more control agents; and instructions for the use of the binding polypeptides, and agents in the diagnosis of a disorder associated with aberrant expression of a DOS molecule. In the preferred embodiments, the binding polypeptides are antibodies or antigen-binding fragments thereof, such as those described above. In these and other embodiments, certain of the binding polypeptides bind to the Mutant DOS protein but do not bind to the DOS protein to further distinguish the expression of these proteins in a biological sample. The invention also provides treatment methods. In general, the treatment methods involve administering an agent to increase expression of a DOS molecule and/or reduce expression of a Mutant DOS molecule. Thus, these methods include gene therapy applications. In certain embodiments, the method for treating a subject with a disorder characterized by aberrant expression of a DOS molecule, involves administering to the subject an effective amount of a DOS nucleic acid molecule to treat the disorder. In yet other embodiments, the method for treatment involves administering to the subject an effective amount of an anti-sense molecule to inhibit (reduce/eliminate) expression of a Mutant DOS nucleic acid molecule and, thereby, treat the disorder. An exemplary molecule for inhibiting expression of a Mutant DOS nucleic acid molecule is an anti-sense molecule that is selective for the mutant nucleic acid and that does not inhibit expression of the DOS nucleic acid molecule. Alternatively, the method for treating a subject with a disorder characterized by aberrant expression of a DOS molecule involves administering to the subject an effective amount of a DOS protein to treat the disorder. In yet another embodiment, the treatment method involves administering to the subject an effective amount of a binding polypeptide to inhibit a Mutant DOS protein and, thereby, treat the disorder. In certain preferred embodiments, the binding polypeptide is an antibody or an antigen-binding fragment thereof; more preferably, the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents The invention provides various research methods and compositions. Thus, according to one aspect of the invention, a method for producing a DOS protein is provided. The method involves providing a DOS nucleic acid molecule operably linked to a promoter, wherein the DOS nucleic acid molecule encodes the DOS protein or a fragment thereof; expressing the DOS nucleic acid molecule in an expression system; and isolating the DOS protein or a fragment thereof from the expression system. Preferably, the DOS nucleic acid molecule has SEQ ID NO:1 or SEQ ID NO:3. According to yet another aspect of the invention, a method for producing a Mutant DOS protein is provided. This method involves: providing a Mutant DOS nucleic acid molecule operably linked to a promoter, wherein the Mutant DOS nucleic acid molecule encodes the Mutant DOS protein or a fragment thereof; expressing the Mutant DOS nucleic acid molecule in an expression system; and isolating the Mutant DOS protein or a fragment thereof from the expression system. Preferably, the Mutant DOS nucleic acid molecule has SEQ ID NO:1 or SEQ ID NO:3, with one or more point mutations or deletions to encode a Mutant DOS protein. These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments. detailed-description description="Detailed Description" end="lead"? |
Video coding method |
The invention relates to a video coding method based on a subband coding scheme applied to a sequence of two-dimensional frames. Said method comprises a subband decomposition step of a current frame (F2), a motion prediction step, carried out with respect to a previous or reference frame (F1), and a coding step. According to the invention, the motion prediction step is based on a redundant decomposition of the reference frame, according to the following procedure: in the approximation subband, the motion is estimated by means of a full search operation applied to each pixel of the current frame, and for the other subbands, the motion is estimated by means of a pel-recursive motion estimation algorithm using for the prediction the reference redundant subband reconstructed from the subsampled subbands. Said pel-recursive motion estimation algorithm itself comprises an initialization sub-step, a determination and updating sub-step, and a decision sub-step. |
1. A video coding method based on a subband coding scheme applied to a sequence of two-dimensional frames, said method comprising a subband decomposition step of a current frame (F2), a motion prediction step, carried out with respect to a previous or reference frame (F1), and a coding step, said method being moreover characterized in that said motion prediction step is based on a redundant decomposition of the reference frame, according to the following procedure: (1) in the approximation subband of said decomposition, the motion is estimated by means of a full search operation involving said reference frame and applied to each picture element (pixel) of the current frame; (2) for the other subbands, the motion is estimated by means of a pixel (or pel) recursive motion estimation algorithm using for the prediction the reference redundant subband reconstructed from the subsampled subbands yielded by said subband decomposition. 2. A coding method according to claim 1, in which said pel-recursive motion estimation algorithm, intended to compute the optic flow between two matrices A and B representing two successive subbands i.e. to estimate for each pixel m its motion vector d(m) in such a manner that B(m)=A(m−d), itself comprises the following sub-steps: (a) an initialization sub-step, the iterative algorithm being initialized with the mean value d0 (m) of the motion vector in a causal neighbourhood S: d 0 ( m ) = 1 Card ( S ) ∑ j ∈ S d ( m - j ) the following notations being used m=pixel for which the motion has to be estimated; d(m)=its motion vector; (b) an updating sub-step, for updating di according to the relation: di+1=di+ui, where ui, determined at each iteration i, is an update vector given by: u i = - E ( m ) ∇ A 2 + λ · ∇ A where ∇A is the gradient of the image at the point m−di, E(m)=B(m)−A(m−di) is the prediction error at the current iteration, and λ is a regularization parameter; (c) a decision sub-step, for carrying a break test and thus ending the procedure if di is close enough to d. 3. A coding method according to claim 2, in which a typical neighbourhood of four pixels is used. 4. A coding method according to claim 2, in which said break test is based on the estimation of the following ratio: R = u i 2 d i 2 said ratio having to be greater than a given threshold ε. 5. A coding method according to claim 4, in which said threshold ε is turned to balance the vector determination precision and the computational load. 6. A coding method according to claim 4, in which said break test includes an additional condition put on a maximum number of iterations. 7. A coding method according to claim 4, in which the determination of said update vector ui is based on a computation step including the following minimizing operation: knowing that B(m)=A(m−di)−δdT.∇A where δdT.∇A is the inner product of ∇A and vector δd=d−di, to minimize the square error J: J=((B(m)−A(m−di)+δdT.∇A)2+λ||δd||2 with respect to δd, the term λ||δd||2 being a regularization term provided in view of a trade-off between the smoothness of the resulting motion vector field, for large values of λ, and the accuracy of the motion vectors, for small values of λ, the minimum being obtained for ∂J/∂δd=0 and leading to the update vector: δ d = u i = - E ( m ) ∇ A 2 + λ · ∇ A 8. A coding method according to claim 7, in which the scanning order is carried out in the following way:even lines from the left to the right, odd lines from the right to the left, the initialization neighbourhood being then also modified for the first line, where it is reduced to the previous pixel, and for the odd lines, where it is inverted to preserve causality 9. A coding method according to claim 7, in which said motion estimation algorithm is re-initialized by testing a break condition with some tolerance, the prediction error when using the estimated motion vector being, for said test, compared to the prediction error with no motion estimation, plus a threshold. 10. A coding method according to claim 2, in which the initialization of the motion vector is done with a linear combination of the average over the neighbourhood S and the motion vector obtained from the subband with the same orientation, at the previous decomposition level. 11. A coding method according to claim 1, in which said pel-recursive motion estimation algorithm, intended to use the correlation that exists between the subbands of a same level of decomposition i.e. to estimate for a given level of decomposition jε{1, 2, . . . , jmax} and for each pixel m its motion vector dj(m) in such a manner that Bj,s(m)=Aj,s(m−dj) for s={0, . . . , 4}, the two matrices A and B representing two successive subbands, itself comprises the following sub-steps: (a) an initialization sub-step, the iterative algorithm being initialized with the mean value dj (m) of the motion vector in a causal neighbourhood S: d j , o ( m ) = 1 Card ( S ) ∑ j ∈ S d j ( m - k ) the following notations being used: kεs(1 to 4); m=pixel for which the motion has to be estimated; dj,o(m)=its motion vector; (b) an updating sub-step, for updating dj,i+1 according to the relation: dj, i+1=dj,i+uj,i, where uj,i determined at each iteration i is an update vector given by: u j , i = ( ∑ s = 0 , 1 3 ( ∇ A j , s ∇ A j , s T ) + λ I ) - 1 ( - ∑ s = 0 , 1 3 E j , s ( m ) ∇ A j , s ) where : ∇ A j , s = ( ∂ A j , s ( m - d j , i ) ∂ m ∂ A j , s ( m - d j , i ) ∂ n ) is the gradient of the image at point m−dj,i and: Ej,s(m)=Bj,s(m)−Aj,s(m−dj,i) (c) a decision sub-step, for carrying a break test and thus ending the procedure if dj,i is close enough to dj. 12. A coding method according to claim 11, in which a typical neighbourhood of four pixels is used. 13. A coding method according to claim 11, in which said break test is based on the estimation of the following ratio: R i , j = u j , i 2 d j , i 2 said ratio having to be greater than a given threshold ε. 14. A coding method according to claim 13, in which said break test includes an additional condition put on a maximum number of iterations. 15. A coding method according to claim 13, in which the determination of said update vector ui is based on a computation step including the following minimizing operation: knowing that Bj,s=Aj,s(m−dj,i)−δdjT.∇Aj,s where δdT.∇A is the inner product of ∇Aj,s and the vector δdj=dj−dj,i, to minimize the square error: min δ dj ( ∑ s = 0 , 1 3 ( B j , s ( m ) - A j , s ( m - d j , i ) + δ j T ∇ A j , s ) 2 + λ δ d j 2 ︸ Ji ) ( 27 ) with respect to δdj, the term λ||δdj||2 being a regularization term provided in view of a trade-off between the smoothness of the resulting motion vector field, for large values of λ, and the accuracy of the motion vectors, for small values of λ, and the minimum being obtained for ∂Jj/∂δdj=0. |
<SOH> BACKGROUND OF THE INVENTION <EOH>In the multimedia domain, new interactive applications such as Internet video streaming, video database browsing or multi-quality video services, are becoming widespread. This recent expansion of video services over heterogeneous networks (Internet, mobile nets, In Home Digital Networks) has raised new issues in terms of varying transport conditions (bandwidth, error rate . . . ) as well as varying consumer demands and terminal decoding capacibilities (CPU, display size, application . . . ), and has led to investigate new algorithms for video compression, in particular methods based on subband decompositions. In the conventional video coding algorithms, and more particularly within the frame of the MPEG-4 standard, the motion estimation between successive frames of a processed video sequence is carried out by means of the so-called block-matching algorithm (BMA):in BMA, a motion vector is assigned to a block of picture elements (pixels) in a current frame (decomposed into blocks of fixed size) by searching a similar block within a defined area—or search window—of a reference frame, the best vector (that represents the shifting of the block) being found by evaluating candidates according to an error measure (the best corresponding block is the one that gives rise to a minimal error). The BMA unfortunately generates high frequency artifacts at the blocks edges (blocking effects) in the motion compensated frames. When the BMA is used in coding schemes based on a wavelet decomposition, these artefacts reduce the coding efficiency by inducing high coefficients in the wavelet decomposition of the compensated frames. |
<SOH> SUMMARY OF THE INVENTION <EOH>The object of the invention is to propose a video coding method incorporating another type of motion estimation and compensation and allowing to improve the coding efficiency. To this end, the invention relates to a method such as defined in the introductory paragraph of the description and which is moreover characterized in that said motion prediction step is based on a redundant decomposition of the reference frame, according to the following procedure: (1) in the approximation subband of said decomposition, the motion is estimated by means of a full search operation involving said reference frame and applied to each picture element (pixel) of the current frame; (2) for the other subbands, the motion is estimated by means of a pixel (or pel) recursive motion estimation algorithm using for the prediction the reference redundant subband reconstructed from the subsampled subbands yielded by said subband decomposition. The proposed technical solution is based on the implementation of a pel-recursive motion estimation algorithm using wavelets. A motion estimation in the wavelet domain has to cope with the problem of translation invariance. The redundant decomposition of the reference frame is then used to predict the motion, allowing thus to take into account motion on the finest resolution grill. In the approximation subband, the motion is estimated by a full search algorithm applied to every pixel in the current frame. For the other subbands, a pel-recursive algorithm using for prediction the reference redundant subband is implemented. The initialization of this algorithm is made using a weighted value of the motion vectors of the spatial neighbours in the same subband and of the motion vector corresponding to the same position at the previous level of the decomposition. The algorithm allows to do a re-initialization at the positions failing to converge towards a good estimate. The scanning order in the subbands is also optimized in order to minimize the drift that can occur in a line-by-line scan. More specifically, said pel-recursive motion estimation algorithm may comprise the following sub-steps: (a) an initialization sub-step, the iterative algorithm being initialized with the mean value d 0 (m) of the motion vector in a causal neighbourhood S: d 0 ( m ) = 1 Card ( S ) ∑ j ∈ S d ( m - j ) the following notations being used: m=pixel for which the motion has to be estimated d(m)=its motion vector; (b) an updating sub-step, for updating di according to the relation: d i+1 =d i +u i , where u i , determined at each iteration i, is an update vector given by: u i = - E ( m ) ∇ A 2 + λ · ∇ A where ∇A is the gradient of the image at the point m−d i , E(m)=B(m)−A(m−d i ) is the prediction error at the current iteration, and λ is a regularization parameter; (c) a decision sub-step, for carrying a break test and thus ending the procedure if d i is close enough to d; a typical neighbourhood of four pixels being preferably used. In a particular implementation of the invention, the break test may be based on the estimation of the following ratio: R = u i 2 d i 2 said ratio having to be greater than a given threshold ε. Said break test may also include an additional condition put on a maximum number of iterations. It may also be indicated that, according to an advantageous embodiment of the invention, the determination of said update vector u i is based on a computation step including the following minimizing operation: knowing that B(m)=A(m−d i )−δd T .∇A where δd T .∇A is the inner product of ∇A and vector δd=d−d i , to minimize the square error J: in-line-formulae description="In-line Formulae" end="lead"? J =( B ( m )− A ( m−d i )+δ d T .∇A ) 2 +λ||δd|| 2 in-line-formulae description="In-line Formulae" end="tail"? with respect to δd, the term λ||δd|| 2 being a regularization term provided in view of a trade-off between the smoothness of the resulting motion vector field, for large values of λ, and the accuracy of the motion vectors, for small values of λ, the minimum being obtained for ∂J/∂δd=0 and leading to the update vector: δ d = u i = - E ( m ) ∇ A 2 + λ · ∇ A In another embodiment of the invention, the motion estimation algorithm may also comprise the following sub-steps: (a) an initialization sub-step, the iterative algorithm being initialized with the mean value d j,o (m) of the motion vector in a causal neighbourhood S: d j , o ( m ) = 1 Card ( S ) ∑ j ∈ S d j ( m - k ) the following notations being used: kεs (1 to 4) m=pixel for which the motion has to be estimated d j,o (m)=its motion vector; (b) an updating sub-step, for updating d j,i+1 according to the relation: in-line-formulae description="In-line Formulae" end="lead"? d i,i+1 =d j,i +u j,i , in-line-formulae description="In-line Formulae" end="tail"? where u j,i determined at each iteration i is an update vector given by: u j , i = ( ∑ s = 0 , 1 3 ( ∇ A j , s ∇ A j , s T ) + λ I ) - 1 ( - ∑ s = 0 , 1 3 E j , s ( m ) ∇ A j , s ) where : ∇ A j , s = ( ∂ A j , s ( m - d j , i ) ∂ m ∂ A j , s ( m - d j , i ) ∂ n ) is the gradient of the image at point m−d j,i and in-line-formulae description="In-line Formulae" end="lead"? E j,s ( m )= B j,s ( m )− A j,s ( m−d j,i ) in-line-formulae description="In-line Formulae" end="tail"? (c) a decision sub-step, for carrying a break test and thus ending the procedure if d j,i is close enough to d j . |
Identification and use of conserved noncoding sequences |
The invention is directed to the identification of conserved noncoding sequences in various organisms and the use of those sequences as primers for use in amplifying, mapping and analysis of DNA among different species. |
1. A method of identifying a polymorphism between two or more DNA sequences; comprising: a) selecting a first and a second primer for use in an amplification reaction for each DNA sequence wherein said first or second primer is a CNS in said two or more DNA sequences; b) hybridizing said first and second primers to each DNA sequence to form primer-DNA hybrids; c) amplifying said primer-DNA hybrids to form amplified DNA products; and d) separating said DNA products by size to identify a polymorphism between said two or more DNA sequences. 2. The method of claim 1 wherein said DNA sequences are plant DNA sequences. 3. The method of claim 1 wherein said polymorphism is utilized for mapping. 4. The method of claim 1 wherein said first primer is a CNS and said second primer is an exon sequence. 5. The method of claim 1 wherein both the first and second primers are CNSS. 6. The method of claim 1 wherein said DNA products are digested with a restriction endonuclease prior to separation in step d). 7. The method of claim 1 wherein said DNA products are separated by electrophoresis. 8. The method of claim 1 wherein said primers are PCR primers. 9. The method of claim 1 wherein the primer-DNA hybrids are amplified using an amplification technique selected from the group consisting of cloning, PCR, LCR, TAS, 3SR, NASBA and QP amplification. 10. The method of claim 9 wherein said technique is PCR. 11. A method of mapping a polymorphism between two or more species; comprising: a) selecting a first and a second primer for use in an amplification reaction wherein said first or second primer is a CNS in the genomes of said two or more species wherein said genomes include genomic DNA; b) hybridizing said first and second primers to said genomic DNA sequences to form primer-genomic DNA hybrids; c) amplifying said primer-genomic DNA hybrids to form amplified DNA products; and d) separating said DNA products by size to map a polymorphism between said two or more DNA sequences. 12. The method of claim 11 wherein said DNA sequences are plant DNA sequences. 13. The method of claim 11 wherein said plant DNA is grass DNA. 14. The method of claim 11 wherein said first primer is a CNS and said second primer is an exon sequence. 15. The method of claim 11 wherein both the first and second primers are CNSs. 16. The method of claim 11 wherein said DNA products are digested with a restriction endonuclease prior to separation in step d). 17. The method of claim 11 wherein said DNA products are separated by electrophoresis. 18. The method of claim 11 wherein said primers are PCR primers. 19. The method of claim 1 wherein the primer-DNA hybrids are amplified using an amplification technique selected from the group consisting of cloning, PCR, LCR, TAS, 3SR, NASBA and Qβ amplification. 20. The method of claim 19 wherein said technique is PCR. |
<SOH> BACKGROUND OF THE INVENTION <EOH>While much progress has been made in recent years in traditional molecular and genetic mapping, sequencing of genomes and molecular analysis of gene expression, there is still a tremendous need to develop improved techniques for molecular and genetic analysis within and between species. Molecular markers are common tools that can reveal polymorphism directly at the DNA level and are used for genetic resource assessment, molecular analysis and genetic mapping. Various types of markers have been developed: RFLP: restriction fragment length polymorphism; PCR: polymerase chain reaction based markers; SCAR: sequence characterized amplified region; SSR: simple sequence repeats (microsatellites); ISSR: intersimple sequence repeats; STS: sequence tagged sites and AFLP: amplified fragment length polymorphisms. Although these methods are powerful, they are useful only within one species or genus because the markers are not from genes shared by larger taxonomic groups. There is thus a need in the art to develop improved methods of genetic mapping and molecular analysis within and across different kingdoms. |
<SOH> SUMMARY OF THE INVENTION <EOH>In order to meet these needs, the present invention is directed to CNSs (conserved noncoding sequences) and their use in molecular analysis and genetic mapping in any organism. The conserved noncoding sequences (CNSs) of the invention find many applications in molecular analysis and genetic mapping. In particular, CNSs find use in identifying polymorphisms between two or more DNA sequences. CNSs find further use in mapping including cross-species alignment of DNA sequences and alignment of syntenic regions outside of coding regions. CNSs may also be utilized in gene cloning including capturing or isolating of sets of related genes. CNSs may be also utilized for analyzing promoter sequences including (a) identifying functional cis-acting sequences within a promoter; (b) identifying binding sites for transcription factor complexes; (c) isolating or physically capturing DNA binding proteins/transcription factors; (d) linking binding sites to appropriate transcription factors via gene expression profiles; (e) designing promoters with novel expression characteristics and (f) titrating out transcription factors via replication of CNS-containing DNA. The CNSs of the invention can also be utilized to alter the expression level and specificity of endogenous genes by in situ modification of CNSs. In one format of the invention, combinations of CNSs or CNS-exon primer combinations are used to generate PCR fragments including highly polymorphic regions and some exon regions. Such a fragment, once shown to be linked with a characteristic of interest in any species, (e.g. any grass) can be sequenced. The sequence, in turn, is used to anchor the linked gene to the nearest species genetic map. The present invention is further directed to methods of genetic mapping using the CNSs of the invention. The present invention is further directed to kits containing various CNSs for use in mapping such CNSS. The present invention is further directed to a method of identifying a polymorphism between two or more DNA sequences by a) selecting a first and a second primer for use in an amplification reaction wherein the first or second primer is a CNS in the two or more DNA sequences; b) hybridizing the first and second primers to the two or more DNA sequences to form primer-DNA hybrids; c) amplifying the primer-DNA hybrids to form amplified DNA products; and d) separating the DNA products by size to identify a polymorphism between said two or more DNA sequences. The DNA sequences may be from any organism including plants. In one format of the method of the invention, the polymorphism is utilized for mapping. In the method of detecting a polymorphism of the invention, the first primer may be a CNS and the second primer may be an exon sequence. Alternatively, both primers may be CNSs. In the method of detecting a polymorphism of the invention the DNA products may be digested with a restriction endonuclease prior to separation in step d). After digestion, the DNA products may be separated by electrophoresis. In one format of the invention, the amplification primers are PCR primers. The primer-DNA hybrids may be amplified using any amplification technique. Such amplification techniques include cloning, PCR, LCR, TAS, 3SR, NASBA and Qβ amplification. The present invention is further directed to a method of mapping a polymorphism between two or more species by a) selecting a first and a second primer for use in an amplification reaction wherein the first or second primer is a CNS in the genomes of the two or more species wherein the genomes include genomic DNA; b) hybridizing the first and second primers to the genomic DNA to form primer-genomic DNA hybrids; c) amplifying the primer-genomic DNA hybrids to form amplified DNA products; and d) separating the DNA products by size to map a polymorphism between the two or more DNA sequences. The present invention is further directed to kits including the CNSs of the invention where the kits find use in detecting polymorphisms and mapping. The kits may include all the components necessary to carry out an amplification reaction utilizing the CNSs. Such components may include primers, enzymes, directions for use, etc. The present invention is further directed to a method of identifying cis-acting genomic DNA sequences that alter gene expression in plants by a) obtaining the genomic DNA sequences of orthologous genes from two or more related plants; b) comparing the non-coding regions of the genomic DNA sequences to identify conserved non-coding sequences at least 7 nucleotides in length and c) testing the CNS in a plant to determine whether the CNS alters gene expression in the plant to thereby identify the CNS as a cis-acting genomic DNA sequences that alters gene expression in plants. In the method of identifying cis-acting genomic DNA sequences that alter gene expression in plants, the genomic DNA sequence of the orthologous genes may be obtained by searching DNA sequence databases or by directly sequencing the genomic DNA. In the method of identifying cis-acting genomic DNA sequences that alter gene expression of the invention the testing of the CNS in a plant may include the steps of: i) creating a CNS-reporter gene construct; ii) introducing the CNS-reporter gene construct into a plant to create a transgenic plant and iii) measuring expression levels of the reporter gene in the transgenic plant. The present invention is further directed to a method of identifying a binding site for transcription factor in a plants, by a) obtaining the genomic DNA sequences of orthologous genes from two or more related plants; b) comparing the non-coding regions of the genomic DNA sequences to identify conserved non-coding sequences at least 7 nucleotides in length and c) testing the CNS to determine whether the CNS is a binding site for a transcription factor complex. In the method of identifying a binding site for a transcription factor of the invention, the genomic DNA sequence of the orthologous genes may be obtained by searching DNA sequence databases or by directly sequencing the genomic DNA. In the method of identifying a binding site for a transcription factor of the invention, the CNS may be tested by a gel shift-binding assay. The present invention is further directed to a method of identifying a conserved noncoding sequence in the genomes of at least two plants by a) identifying coding regions in the genomes; b) masking out the coding regions to formed masked genomic sequences and unmasked genomic sequences in said genomes; c) comparing the unmasked genomic sequences to identify two or more conserved noncoding sequences wherein the conserved noncoding sequences contain at least 7 identical nucleotides to identify the conserved non-coding sequences. In the method of identifying a conserved noncoding sequence of the invention, the unmasked genomic sequences may be compared using a word size of seven, a gap penalty existence from 2-5 and a gap penalty extension from 1-2. The method of identifying a conserved noncoding sequence may further include identifying the position of the conserved noncoding sequences relative to the masked genomic sequence. |
Termminal providing system |
A plurality of terminals (40) that are provided to users, and a providing device (20) are placed at a providing place (2). The providing device includes a memory that stores information concerning the plurality of terminals (40). The providing device (20) specifies a terminal (40) that includes the application software that the user designates from the plurality of terminals (40) by applying information in the memory, and provides the terminal (40) to the user. A collecting device (30) is placed at a collecting place (3) for collecting terminals that are returned by the users, and collects terminals (40) returned by the users. |
1-34. (Canceled) 35: A terminal providing system comprising: a plurality of terminals placed at a providing place for providing terminals to users, and comprising one or more application software; a providing device placed at the providing place, and that carries out processing for providing to a user, a terminal which comprises the application software that the user designates, from the plurality of terminals; and a collecting device placed at a collecting place for collecting the terminals provided to the users, and that carries out processing for collecting terminals which are returned by the users; wherein the providing device comprises a memory which stores information concerning the plurality of terminals which are placed at the providing place, a specifying unit which specifies a terminal which comprises the application software that the user designated, applying the information that said memory stores, and a presenting unit which presents to the user, the terminal specified by the specifying unit. 36 A terminal providing system according to claim 25, wherein said collecting device comprises: a determining unit which determines whether the user who received providing of the terminal and the user who returns the terminal is the same; a collecting unit that carries out processing for collecting the terminal, and stops the processing for collecting the terminal in a case where it is determined that the user is the same, and stops the processing for collecting the terminal in a case where it is determined that the user is not the same. 37: The terminal providing system according to claim 35, wherein said collecting device comprises a deleting unit which deletes data that is stored in the returned terminal. 38: The terminal providing system according to claim 35, further comprising an output device that is provided at the collecting place, and that outputs data that is stored in the returned terminal, wherein data stored in the terminal is possible to be output only by the output device. 39: The terminal providing system according to claim 38, wherein said output device comprises at least one of a printer that prints data to paper, a recorder that records data to a recording medium, and a sending device that sends data to external terminals by a communication network. 40: The terminal providing system according to claim 39, further comprising a charging device which charges the user according to an amount of data output by the output device. 41: The terminal providing system according to claim 35, further comprising a management device which manages the providing of the plurality of terminals, wherein the management device comprises a reservation reception unit which receives reservation of terminals. 42: The management system according to claim 41, wherein said management device comprises a charging unit which obtains a utility fee of the terminal, based on at least one of the model of terminal, a kind of application software the terminal has, and a utility time of the terminal. 43: A terminal providing system comprising: a plurality of terminals placed to each of a plurality of providing/collecting places, which are provided to a plurality of stations of vehicles, for providing terminals to users and collecting the terminals that are returned by the users, and that include one or more application software; and a plurality of providing/collecting devices placed at the plurality of providing/collecting places; wherein each of the plurality of providing/collecting devices comprises a providing unit which carries out processing for providing to a user, a terminal that has the application software that the user designates, and a collecting unit which carries out processing for collecting the terminal that is returned from the user. 44: The terminal providing system according to claim 43, wherein said providing/collecting device comprises a determining unit which determines whether the user who is provided the terminal is the same as the user who is returning the terminal, and the collecting unit may carry out processing for collecting the terminal in a case where the determining unit determines that the user is the same, and may stop the processing for collecting the terminal in a case where the determining unit determines that the user is not the same. 45: The terminal providing system according to claim 43, wherein said providing/collecting device comprises a deleting unit which deletes data stored in the returned terminal. 46: The terminal providing system according to claim 43, further comprising a plurality of output devices placed at the plurality of providing/collecting places, and which output data that are stored in the returned terminals, wherein data stored in the terminal is possible to be output, only by the plurality of output devices. 47: The terminal providing system according to claim 46, wherein each of the output devices comprises at least one of a printer that prints data to paper, a recorder that records data to a recording medium, and a sending device that sends data to external terminals by a communication network. 48: The terminal providing system according to claim 46, further comprising a charging device which charges the user of a utility fee in accordance with an amount of data output by the plurality of output devices. 49: The terminal providing system according to claim 43, further comprising a management device which manages the providing of the plurality of terminals, wherein the management device comprises a reservation reception unit which receives reservation of terminals. 50: The terminal providing system according to claim 49, wherein said management device comprises a first specifying unit which specifies a terminal that has the application software that the user designates, from the terminals which are placed at the station where the user is to ride a vehicle, wherein the reservation reception unit receives reservation of the terminal specified by said first specifying unit. 51: The terminal providing system according to claim 50, wherein the management device comprises a time and date obtaining unit which obtains a possible time and date when the terminal provided to the user becomes possible to be provided to another user, by applying a time table that shows arrival and departure time of each station of the vehicles. 52: The terminal providing system according to claim 51, wherein said management device comprises a second specifying unit which specifies terminals in which the possible time and date is before the time and date that the user rides the vehicle, from the terminals placed at the station where the user is to ride the vehicle, wherein the first specifying unit specifies the terminal that comprises the application software that the user designated, from the terminals specified by the second specifying unit. 53: A terminal providing system according to claim 49, wherein said management device comprises a charging unit which obtains a utility fee of the terminal, based on at least one of a model of terminal, a kind of application software the terminal has, and a utility time of the terminal. 54: The terminal providing system according to claim 48, further comprising an electric power unit provided at a seat of the vehicle, and that provides electric power to the terminal, wherein said terminal is possible to be provided electric power only from said electric power unit. 55: The terminal providing system according to claim 54, wherein said electric power unit comprises an indicator which indicates a remaining amount of electric power that is possible to be provided. 56: The terminal providing system according to claim 54, wherein said electric power unit provides electric power to said terminal, in accordance with an amount of money paid by the user. 57: A terminal providing device provided at a providing place for providing terminals to users, and which carries out processing for providing terminals to users and comprises: a memory which stores information concerning a plurality of terminals which comprise one or more application software; and a specifying unit which specifies one of said terminals which comprises an application software that is designated by a user, by applying information that said memory stores; and a presenting unit which presents to the user the terminal specified by the specifying unit. 58: A terminal management device placed in each of a plurality of providing/collecting places provided at a plurality of stations of vehicles, to provide terminals to users and collect terminals returned by the users, which manages the providing of the terminals which comprise one or more application software, and comprises: a first specifying unit which specifies one of the terminals that comprise an application software that a user designated from the terminals that are placed at stations where the user rides the vehicle; and a reservation receiving unit which receives reservation of the terminal that is specified by said first specifying unit. 59: The terminal management device according to claim 58, further comprising a time and date obtaining unit which obtains a possible time and date when the terminal provided to the user becomes possible to be provided to another user, by applying a time table that shows arrival and departure time of each station of the vehicles. 60: The terminal management device according to claim 59, further comprising a second specifying unit which specifies terminals in which said possible time and date is before the time and date that the user rides the vehicle, from the terminals placed at the station where the user is to ride the vehicle, wherein said first specifying unit specifies the terminal that comprises the application software that the user designated, from the terminals specified by the second specifying unit. 61: A terminal providing method comprising: a providing which carries out processing for providing a terminal to a user; and a collecting which carries out processing for collecting the terminal that is retuned by the user, wherein said providing comprises a specifying of specifying a terminal which includes an application software designated by a user, from the plurality of terminals which can be provided to users, and comprise one or more application software, and a presenting of the specified terminal to the user. 62: The terminal providing method according to claim 61, wherein the plurality of terminals are respectively placed at a plurality of stations of vehicles, wherein said specifying comprises a first specifying the terminal that comprises the application software that is designated by the user, from the terminals that are respectively placed at a plurality of stations where the user rides the vehicle. 63: The terminal providing method according to claim 62, further comprising a time and date obtaining of obtaining a possible time and date when the terminal provided to the user becomes possible to be provided to another user, by applying a time table that shows arrival and departure time of each station of the vehicles. 64: The terminal providing method according to claim 63, wherein the specifying comprises a second specifying terminals in which said possible time and date is before the time and date that the user rides the vehicle, from the terminals placed at the station where the user is to ride the vehicle, wherein said first specifying comprises specifying the terminal that comprises the application software that the user designated, from the terminals specified by the second specifying. 65: A computer readable recording medium that stores a program to function as a terminal providing device provided at a providing place for providing terminals to users, and which carries out processing for providing terminals to users, and comprises: a memory which stores information concerning a plurality of terminals which comprise one or more application software; and a specifying unit which specifies the terminal which comprises an application software that is designated by a user, by applying information that said memory stores; and a presenting unit which presents the terminal specified by said specifying unit. 66: A computer readable recording medium that stores a program to function as a terminal providing device which manages a providing situation of a plurality of terminals including one or more application software, which are placed at each of a plurality of providing/collecting places, which are provided to a plurality of stations of vehicles, for providing terminals to users and collecting the terminals that are returned by the users, and comprises: a first specifying unit which specifies a terminal that has an application software that a user designates, from the terminals that are placed at a station where the user is to ride a vehicle; and a reservation reception unit which receives reservation of the terminal specified by the first specifying unit. 67: A program to function a computer as a terminal providing device provided at a providing place for providing terminals to users, and which carries out processing for providing terminals to users, and comprises: a memory which stores information concerning a plurality of terminals that comprise one or more application software; and a specifying unit which specifies a terminal that comprises an application software that is designated by a user, by applying information that said memory stores; and a presenting unit which presents to the user, the terminal specified by said specifying unit. 68: A program to function a computer as a terminal management device which manages a providing situation of a plurality of terminals including one or more application software, which are placed at each of a plurality of providing/collecting places, which are provided to a plurality of stations of vehicles, for providing terminals to users and collecting the terminals that are returned by the users, and comprises: a first specifying unit which specifies a terminal that has an application software that a user designates, from the terminals that are placed at the station where the user is to ride a vehicle; and a reservation reception unit which receives reservation of the terminal specified by the first specifying unit. |
<SOH> BACKGROUND ART <EOH>In business, etc., there are cases where portable terminals (note book computers, etc.) are applied to create documents, when transferring to a destination, to present the documents at the destination to clients. In this case, even in a case where the portable terminal is used during transferring to the destination, or at the destination, a user has to carry the portable terminal from his/her house or company. In other words, the user has to carry the portable terminal even when the portable terminal is not necessary. Therefore, the object of the present invention is to provide a terminal providing system, a terminal providing device, a terminal management device, a terminal providing method, a recording medium, and a program for providing a user terminal to a user, only when it is necessary for the user. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a structure diagram showing a terminal lending system according to a first embodiment. FIG. 2A is a structure diagram of a management device that structures the terminal lending system in FIG. 1 , FIG. 2B is a diagram showing a management table that is stored by a database that the management device comprises, and FIG. 2C is a diagram showing a user information table that the database stores. FIG. 3A is a structure diagram of a providing terminal that structures the terminal lending system in FIG. 1 , and FIG. 3B is a diagram showing a lending table stored by a storing unit that structures providing terminal. FIG. 4 is a structure diagram of a collecting terminal that structures the terminal lending system in FIG. 1 . FIG. 5 is a flowchart showing a lending processing carried out by a control unit that structures the providing terminal in FIG. 3A . FIG. 6 is a flowchart showing a returning processing carried out by a control unit that structures the collecting terminal in FIG. 4 . FIG. 7 is a structure diagram of a terminal lending system according to a second embodiment. FIG. 8A is a diagram showing a management table that is stored by the database that the management device that structures the terminal lending system in FIG. 7 comprises, FIG. 8B is a diagram showing a reserving person table that the database stores, and FIG. 8C is a diagram showing a lending table that a storing unit of a providing terminal that structures the terminal lending system in FIG. 7 stores. FIG. 9 is a flowchart showing a reservation processing carried out by a control unit of the management device. FIG. 10 is a flowchart showing a lending processing carried out by the control unit of the providing terminal. FIG. 11 is a structure diagram of a terminal lending system according to a third embodiment. FIG. 12A is a diagram showing a management table stored by a database that a management device that structures the terminal lending system in FIG. 11 comprises, and FIG. 12B is a diagram showing a time table that the database stores. FIG. 13A is structure diagram of a providing/collecting terminal that constitutes the terminal lending system in FIG. 11 , and FIG. 13B is a diagram showing an inventory table stored by a storing unit that structures the providing/collecting device. FIG. 14 is a flowchart showing a reservation processing carried out by the control unit of the management device. FIG. 15 is a flowchart showing a lending processing carried out by a control unit of the providing/collecting terminal. FIG. 16 is a diagram showing an example where an electric supply unit is placed at a seat of a vehicle. detailed-description description="Detailed Description" end="lead"? |
Privacy filter |
The invention relates to a method for reversibly rendering at least one object in an image unrecognizable, whereby each object to be rendered unrecognizable is automatically detected in an original image and at least one data set is generated comprising image information for the representation of the image in which each object to be rendered unrecognizable is, in fact, rendered unrecognizable, and additional information that permits the restoration of each object that has been rendered unrecognizable in the image. |
1. A method for reversibly rendering at least one object in an image unrecognizable, comprising the steps of: automatically detecting any object to be rendered unrecognizable in an original image, generating at least one data set comprising: image information for representation of the image in which each object to be rendered unrecognizable is in fact rendered unrecognizable, additional information to be able to restore each object rendered unrecognizable in the image. 2. A method according to claim 1 in which a data set is generated comprising the image information and the additional information. 3. A method according to claim 1 in which at least two data sets are generated, whereby a first data set comprises the image information and a second data set comprises the additional information. 4. A method according to claim 1 in which each object is rendered unrecognizable with the same code. 5. A method according to claim 1 in which at least two objects are rendered unrecognizable with different codes. 6. A method according to claim 1 in which each object of a previously determined object class is automatically detected and rendered unrecognizable. 7. A method to restore at least one object rendered unrecognizable in an image according to a method according to claim 1, comprising the steps of: evaluating the additional information in order to generate a restored partial image information corresponding to the at least one object rendered unrecognizable, replacing a corresponding area of the image information by the restored partial image information. 8. A method according to claim 7 with which, in an image with several objects rendered unrecognizable, individually selectable objects are restorable. 9. A method according to claim 7 with which all objects rendered unrecognizable are restored with at least one specified common characteristic. 10. A method according to claim 7 with which all objects rendered unrecognizable are restored. 11. A method according to claim 2 in which each object is rendered unrecognizable with the same code. 12. A method according to claim 3 in which each object is rendered unrecognizable with the same code. 13. A method according to claim 2 in which at least two objects are rendered unrecognizable with different codes. 14. A method according to claim 3 in which at least two objects are rendered unrecognizable with different codes. 15. A method according to claim 4 in which each object of a previously determined object class is automatically detected and rendered unrecognizable. 16. A method according to claim 5 in which each object of a previously determined object class is automatically detected and rendered unrecognizable. 17. A method to restore at least one object rendered unrecognizable in an image according to a method according to claim 2, comprising the steps of: evaluating the additional information in order to generate a restored partial image information corresponding to the at least one object rendered unrecognizable, replacing a corresponding area of the image information by the restored partial image information. 18. A method to restore at least one object rendered unrecognizable in an image according to a method according to claim 3, comprising the steps of: evaluating the additional information in order to generate a restored partial image information corresponding to the at least one object rendered unrecognizable, replacing a corresponding area of the image information by the restored partial image information. 19. A method to restore at least one object rendered unrecognizable in an image according to a method according to claim 6, comprising the steps of: evaluating the additional information in order to generate a restored partial image information corresponding to the at least one object rendered unrecognizable, replacing a corresponding area of the image information by the restored partial image information. 20. A method according to claim 8 with which all objects rendered unrecognizable are restored with at least one specified common characteristic. |
<SOH> FIELD OF THE INVENTION <EOH>The invention relates to an automatic method for reversibly rendering objects in static and moving images unrecognizable, especially of people and faces, and a method for the restoration of selected objects that have been rendered unrecognizable. |
Health feed |
The present invention relates to a method of promoting immunoglobulin A secretion in the mucosal membranes of non-human animals. The method comprises administering a foodstuff comprising glutamine to the non-human animal. |
1. A method of promoting immunoglobulin A secretion in a mucosal membrane of a non-human animal comprising administering a foodstuff comprising glutamine to the non-human animal. 2. The method of claim 1, wherein the mucosal membrane is the gastrointestinal tract of the non-human animal. 3. The method of claim 1, wherein in the mucosal membrane is the urogenital tract of the non-human animal. 4. A method as claimed in claim 1 for the use of maintaining or improving the health of a non-human animal. 5. The method of claim 4, wherein the non-human animal is healthy. 6. The method of claim 1, wherein the non-human animal is a companion animal selected from the group consisting of a cat and a dog, or wherein the animal is a porcine, ovine, bovine or poultry animal. 7. The method of claim 1, wherein glutamine is one or more of L-glutamine, a peptide comprising glutamine or an extract comprising glutamine. 8. The method of claim 7, wherein the peptide is one or more of a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, a longer chain peptide or a peptide mixture. 9. The method of claim 8, wherein the peptide mixture is derived from one or more of gliadin, oat bran, soya bean meal, linseed, cereals, forages, sunflower, lupin, beans, lentils, milk powder, caesin, whey, soy casein hydrosylate or wheat gluten. 10. The method of claim 1, wherein glutamine is provided to the foodstuff at a level of approximately 1% w/w on a dry matter basis or above. 11. The method of claim 1, wherein the foodstuff contains from approximately 3% to approximately 15% moisture. 12. The method as of claim 1, wherein glutamine is provided to the non-human animal at a level of approximately 0.1 g per kilogram body weight or above per day. 13. The method as of claim 1, wherein the foodstuff is provided one or more times per day. 14. A method of producing a composition for preventing or treating infection in the gastrointestinal tract of a non-human animal comprising the step of providing glutamine to a foodstuff. 15. A method of producing a composition for promoting urogenital health in a non-human animal comprising the step of providing glutamine to a foodstuff. 16. The method of claim 15, wherein the composition prevents or treats infection in the urogenital tract of a non-human animal. 17. The method claim 14, wherein the infection is caused by a virus or a bacteria. 18. The method of claim 14, wherein the non-human animal is healthy. 19. The method of claim 14, wherein the non-human animal is a companion animal selected from the group consisting of a cat and a dog or wherein the animal is a porcine, ovine, bovine or poultry animal. 20. The method of claim 19, wherein glutamine is one or more of L-glutamine, a peptide comprising glutamine or an extract comprising glutamine. 21. The method of claim 20, wherein the peptide is one or more of a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, a longer chain peptide or a peptide mixture. 22. The method of claim 21, wherein the peptide mixture is derived from one or more of gliadin, oat bran, soya bean meal, linseed, cereals, forages, sunflower, lupin, beans, lentils, milk powder, caesin, whey, soy, casein hydrosylate or wheat gluten. 23. The method of claim 14, wherein the glutamine is provided to the composition at a level of approximately 1% w/w on a dry matter basis or above. 24. The method of claim 14, wherein the composition contains from approximately 3%, 4% or 5% to approximately 15% moisture. 25. The method of claim 14, wherein the glutamine is provided to the non-human animal at a level of approximately 0.1 g per kilogram body weight or above per day. 26. The method of claim 14, wherein the composition is provided one or more times per day. 27. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Glutamine is a neutral amino acid, which is readily transported across plasma membranes. As an important intermediate in a number of metabolic pathways, cellular utilization of glutamine can far exceed that of other amino acids especially within intestinal and immune cells. Glutamine is important in the transport of amino nitrogen and ammonia, as a substrate in gluconeogenesis and ammoniagenesis, as a fuel source for rapidly dividing cells and may also be involved in the regulation of protein synthesis. The high rate of glutamine utilization by the intestine (identified to occur in man) may be due in part to the large lymphocyte and macrophage populations in intestinal walls and Peyer's patches. These cells exhibit high glutaminase activity and utilize glutamine as their preferential fuel source, even in the quiescent state. However, as with many other cells that require glutamine, both enterocytes and lymphocytes lack the synthetic apparatus to produce glutamine relying solely on circulatory or dietary sources. It is suggested that a fall in the plasma concentration could compromise lymphocyte function accounting for the increase in susceptibility to viral infection. Gut-associated lymphoid tissue in the gastrointestinal tract appears to provide immunologic protection for the gastrointestinal tract and for extra intestinal mucosal sites such as the nasopharynx, mammary glands, salivary glands and lungs. Lymphocytes, which provide or control the production of immunoglobulin A are released by the gut-associated lymphoid tissue and distributed to the gastrointestinal tract and extra intestinal sites via the mesenteric lymphatic channels and thoracic duct. Feeding of an individual by total parenteral nutrition results in the atrophy of gut-associated lymphoid tissue and a decrease in immunoglobulin A levels. Addition of glutamine to a total parenteral nutrition solution was shown by Li et al to normalize gut-associated lymphoid tissue population. Work by Gismondo et al (Gismondo, M. R., Drago, L., Fassina, M. C., Vaghi, I., Abbiati, R. and Grossi, E. (1998). Immunostimulating effect of oral glutamine. Dig. Dis. Sci, 43, 1752-1754) has indicated that the oral administration of glutamine to congenitally immunosuppressed mice provides a positive effect on serum levels of interleukin 2 and the intestinal population of T cells. In addition, studies in man by Fujita and Sakura (Fujita, T. and Sakurai, K. (1995) Efficacy of glutamine-enriched enteral nutrition in an experimental model of mucosal ulcerative colitis. Br. J. Surg, 82, 749-751) have indicated that the administration of glutamine is therapeutically beneficial to patients with inflammatory bowel disease. It is a desire in the area of pet food products and companion animal health as well as farm animal health to provide diets suitable to support the health of non-human animals. In particular it is desire to provide diets suitable to promote or maintain the health of already healthy non-human animals. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The first aspect of this invention relates to a method of promoting immunoglobulin secretions in the mucosal membranes of a non-human animal comprising administering a foodstuff comprising glutamine to a non-human animal. Mucosal membranes are the moist membranes lining many tubular structures and cavities. These membranes provide a protective layer between the external environment and the internal organs of an animal. Mucosal cells/tissues include mucosal coverings of the gut, the mouth, the nasal passage, the esophagus, the stomach, the lung, the small intestine, the large intestine, epithelial tissue, urogenital tract, the eyes, and mammary glands. The method of the first aspect seeks to improve and maintain the health of a non-human animal. In particular, the animals of the first aspect of the invention are pet or companion animals such as cats or dogs or farm animals such as swine (porcine), sheep (ovine) or cattle (bovine) or poultry. The animals may be at any life-stage, such as young, adult or senior. Accordingly, kittens, puppies, piglets, lambs, calves and chicks are covered by the present invention. The maintenance and improvement of the health of a pet or companion animal and of other animals, such as farm animals is a constantly ongoing aim in the art. It is possible to monitor the improved health of an animal as achieved by the invention in a number of ways. Two of these are feces quality and gastrointestinal (GI) tract health. By improving the health of the animal, the invention seeks to promote and maintain good quality feces in animals (such as pet animals). Good feces quality is of two-fold importance. Firstly, it is a good indicator of a healthy animal (such as a pet). It is known that good feces quality usually reflects healthy colonic structure and function. Secondly, it is a much-favored practicality for pet-owners. The invention also aims to improve the GI tract health of animals (such as pet animals). The ability to maintain and improve GI tract health can be beneficial to animal owners (such as pet owners) because it has an impact on their animal's overall health. Without being bound by scientific theory, it is believed that by increasing the level of secretary immunoglobulin A in the mucosal membranes, glutamine maintains and promotes health of a non-human animal. It is postulated that elevated levels of immunoglobulin A improve the defense mechanisms of the mucosal membranes by eliminating viruses from epithelial cells and by forming a barrier which prevents the adherence of pathogenic bacteria. For example, elevated levels of immunoglobulin A in the lungs and/or the nasopharynx may protect an animal from micro-organisms that cause influenza or pneumonia. Within the gastrointestinal tract, immunoglobulin A is believed to decrease intestinal permeability and to enhance intestinal absorption. Therefore, a preferred feature of the first aspect of the invention relates to a method for increasing levels of secretory immunoglobulin A in the gastrointestinal tract comprising administering a foodstuff comprising glutamine to the non-human animal. A further preferred feature of the first aspect of the invention relates to a method for increasing the levels of secretory immunoglobulin A in the urogenital tract comprising administering a foodstuff comprising glutamine to a non-human animal. It is a preferred feature of the invention that the foodstuff of the first aspect is administered to a non-human animal, which is healthy. It is postulated that administering a foodstuff comprising glutamine to a healthy non-human animal will allow the healthy status of that animal to be maintained, as the animal will be less susceptible to viral or bacterial infection. For the purposes of this invention, ‘health’ is defined as an absence of clinical disease. Thus a healthy animal is an animal which does not exhibit the symptoms of a clinical disease, for example, by its immune status or histology. In a preferred aspect of this invention, the term healthy encompasses animals at optimal health. In another preferred aspect of this invention, the term healthy encompasses animals at optimal or sub optimal health, for example animals with one or more subclinical diseases. An assessment of the health of a particular animal can be carried out by the owner (i.e. by assessing the quality of the feces of the animal and/or monitoring the appetite of the animal) or by an individual qualified to do so (e.g. a veterinary surgeon, dietician) by assessing the histology and/or immune status of the animal. The method of the first aspect comprises administering a foodstuff comprising glutamine. The foodstuff may comprise one or more components which provide a source of glutamine such as one or more of gliadin, oat bran, soya bean meal, linseed, cereals, forages, sunflower, lupin, beans, lentils, milk powder, caesin, whey or soy. For the purposes of this invention, cereals include barley, oats, wheat, bran and rye and forages include grass, hay, rye grass etc. Alternatively, the foodstuff may be supplemented by a source of glutamine. The glutamine source may be the free amino acid (preferably L-glutamine), a peptide rich in L-glutamine or an extract containing L-glutamine. Suitable peptides rich in L-glutamine include dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, longer chain peptides or peptide mixtures. Such peptide mixtures include proteins rich in L-glutamine, hydrolates or fractions thereof (e.g. peptide mixture(s) obtained from one or more of gliadin, oat bran, soya bean meal, linseed, cereals, forages, sunflower, lupin, beans, lentils, milk powder, caesin, whey or soy). Glutamine can be further provided by an extract containing L-glutamine either as a free amino acid or as a peptide containing L-glutamine (e.g. extracts of gliadin, oat bran, soya bean meal, linseed, cereals, forages, sunflower, lupin, beans, lentils, milk powder, caesin, whey or soy). Other L-glutamine derivatives include L-glutamine salts, N-acyl derivatives of L-glutamine including N-alkanoyl L-glutamine compounds such as N-acetyl L-glutamine. The N-acylation of L-glutamine stabilises the peptide, in comparison with free amino acid L-glutamine. Such peptides may be pH and fluid stable. The dipeptide can be but is not limited to L-alanyl-L-glutamine or L-glycyl-L-glutamine. The dipeptide containing L-glutamine should be stable in solution. Preferably, the glutamine is provided as the free amino acid L-glutamine or a dipeptide containing L-glutamine. Alternatively, the glutamine is provided either as an extract or peptide mixture from or by wheat gluten or a casein hydrosylate such as sodium caseinate. Glutamine is preferably provided to the foodstuff at a level of from 0.01% to 10% w/w on a dry matter basis. Preferably the glutamine is provided as free glutamine or a source thereof having an equivalent amount of bioavailable glutamine. Preferably, the glutamine is provided at a level of 0.01% to 5% w/w on a dry matter basis, most preferably, approximately 1% w/w on a dry matter basis or above. The glutamine of the invention would be provided by the foodstuff at levels of approximately 0.01 g to approximately 1 g per kg body weight per day, more preferably, approximately 0.1 g per kg body weight or above per day. The foodstuff is preferably administered daily, more preferably twice daily. Where the foodstuff is a treat or snack, the foodstuff can be administered one or more times a day, preferably five or more times a day. The amount of glutamine in a foodstuff may therefore vary depending on the number of times a day the foodstuff is to be administered. The foodstuff of the invention may be a dry product (with approximately 3, 4 or 5 to approximately 15% moisture), a semi-moist product (with approximately 15 to approximately 70% moisture) or a wet product (with approximately 70 to approximately 90% moisture). The foodstuff according to the present invention encompasses any product that an animal, (such as a pet) consumes in its diet. Thus, the invention covers standard food products as well as food snacks, such as pet food snacks (for example, snack bars, treats, biscuits and sweet products). The foodstuffs preferably a cooked product. It may incorporate meat or animal derived material (such as beef, chicken, turkey, lamb, fish, blood plasma, marrow bone etc or one or more thereof). The product alternatively may be meat free (preferably including a meat substitute such as soya, maize gluten or a soya product) in order to provide a protein source. The product may contain additional protein sources such as soya protein concentrate, milk proteins, gluten etc. The product may also contain a starch source such as one or more grains (e.g. wheat, corn, rice, oats, barley etc), or may be starch free. The foodstuff of the invention is preferably produced as a dry product containing from approximately 3%, 4% or 5% to approximately 15% moisture. The preferred dry food is more preferably presented as small biscuit-like kibbles. The foodstuff is preferably packaged. In this way, the consumer is able to identify, from the packaging, the ingredients in the foodstuff and confirm that it is suitable for the particular animal (such as a pet) in question. The packaging may be metal (usually in the form of a tin or flexifoil), plastic (usually in the form of a pouch), paper or card. The amount of moisture in any product may influence the type of packaging, which can be used or is required. The foodstuff of the first aspect can be provided as a food supplement. The food supplement can be a powder, sauce, topping, biscuit, kibble, pocket or tablet that can be administered with or without an additional foodstuff. Where the food supplement is administered with an additional foodstuff, the food supplement can be administered sequentially simultaneously or separately. The food supplement may mixed with the foodstuff, sprinkled over the foodstuff or served separately. Alternatively, the food supplement can be added to a liquid provided for drinking such as water or milk. The foodstuff can be made according to any method known in the art. Foodstuffs for pet animals can be any, including such as in Waltham Book of Dog and Cat Nutrition, Ed. ATB Edney, Chapter by A. Rainbird, entitled “A Balanced Diet” in pages 57 to 74 Pergamon Press Oxford. The glutamine may be mixed with the other components of the foodstuff or can be added to the completed foodstuff. In a preferred feature of the invention, the glutamine is coated or sprayed on to the surface of the foodstuff. Alternatively, one or more components comprising glutamine are admixed, with one or more other components of the foodstuff. The second aspect of the invention relates to the use of glutamine in the manufacture of a foodstuff for preventing or treating infection in the gastrointestinal tract. It is proposed that the administration of the foodstuff will result in an increase in the level of secretary immunoglobulin A in the gastrointestinal tract. It is postulated that such elevated levels of immunoglobulin A will prevent viral or bacterial infection by eliminating viruses from epithelial cells and forming a barrier which prevents adherence of pathogenic bacteria. In addition, elevated immunoglobulin A levels are believed to decrease intestinal permeability and enhance intestinal absorption. Thus the second aspect of the invention further relates to the use of glutamine in the manufacture of a foodstuff for preventing or treating bacterial or viral infections such as calicivirus in the gastrointestinal tract. All preferred features of the first aspect of the invention also relate to the second aspect. The third aspect of the invention relates to the use of glutamine in the manufacture of a foodstuff for the promotion or maintenance of the urogenital health of a non-human animal. In particular, the third aspect relates to the use of glutamine in the manufacture of a foodstuff for preventing or treating infection in the urogenital tract of a non-human animal. For the purposes of the third aspect, the infections are preferably bacterial or viral infections of the urogenital tract, such as calicivirus. All preferred features of the first and second aspects of the invention also relate to the third aspect. detailed-description description="Detailed Description" end="lead"? |
Novel sulfated saccharide and process for producing the same |
A sulfated saccharide represented by the following general formula (I): [A]-(6SO3-GlcNAc)-[C]—R (I) wherein (6SO3-GlcNAc) represents a 6-sulfated N-acetylglucosamine residue, [A] represents a hydroxyl group or a saccharide residue, [C] represents a saccharide residue and R represents a hydroxyl group or a substituted hydroxyl group is disclosed. The sulfated saccharide effectively bonds to selectin protein which is present in leukocytes and hence has anti-inflammatory activity. The sulfated saccharide is expected to be widely used in the medicinal field, etc. |
1. A sulfated saccharide represented by the following general formula (I): [A]-(6SO3-GlcNAc)-[C]—R (I) wherein (6SO3-GlcNAc) represents a 6-sulfated N-acetylglucosamine residue, [A] represents a hydroxyl group or a saccharide residue, [C] represents a saccharide residue and R represents a hydroxyl group or a substituted hydroxyl group. 2. A sulfated saccharide as recited in claim 1, wherein the saccharide residue represented by [A] of the general formula (I) is a saccharide residue derived from a saccharide selected from glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and oligosaccharides thereof. 3. A sulfated saccharide as recited in claim 1 or 2, wherein the substituted hydroxyl group represented by [C] of the general formula (I) is p-nitrophenoxy group. 4. A sulfated saccharide as recited in any one of claims 1 through 3, wherein the saccharide residue represented by [A] contains a sulfate group or a phosphate group. 5. A sulfated saccharide as recited in any one of claims 1 through 4, wherein the saccharide residue represented by [C] of the general formula (I) is a saccharide residue derived from a saccharide selected from glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and oligosaccharides thereof. 6. A sulfated saccharide as recited in claim 5, wherein the saccharide residue represented by [C] contains a sulfate group or a phosphate group. 7. A sulfated saccharide represented by the following general formula (II): HO-(GlcNAc)-(6SO3-GlcNAc)-[D] (II) wherein (6SO3-GlcNAc) represents a 6-sulfated N-acetylglucosamine residue, (GlcNAc) represents an N-acetylglucosamine residue, and [D] represents a hydroxyl group, a substituted hydroxyl group or a saccharide residue. 8. A sulfated saccharide as recited in claim 7, wherein the saccharide residue represented by [D] of the general formula (II) is a saccharide residue derived from a saccharide selected from glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and oligosaccharides thereof. 9. A sulfated saccharide as recited in claim 7 or 8, wherein the substituted hydroxyl group represented by [D] of the general formula (II) is p-nitrophenoxy group. 10. A sulfated saccharide as recited in any one of claims 7 through 9, wherein the saccharide residue represented by [D] contains a sulfate group or a phosphate group. 11. A process of preparing a sulfated saccharide represented by the following general formula (I): [A]-(6SO3-GlcNAc)-[C]—R (I) wherein (6SO3-GlcNAc) represents a 6-sulfated N-acetylglucosamine residue, [A] represents a hydroxyl group or a saccharide residue, [C] represents a saccharide residue and R represents a hydroxyl group or a substituted hydroxyl group, characterized in that a sulfated saccharide represented by the following general formula (Ia): [A]-(6SO3-GlcNAc)-[B] (Ia) wherein (6SO3-GlcNAc) and [A] have the same meaning as above and [B] represents a hydroxyl group or a substituted hydroxyl group, is reacted with a saccharide of the following general formula (Ib): HO—[C]—R (Ib) wherein [C] represents a saccharide residue and R represents a hydroxyl group or a substituted hydroxyl group, in the presence of N-acetylhexosaminidase or N-acetylglucosaminidase. 12. A process of preparing a sulfated saccharide represented by the following general formula (II): HO-(GlcNAc)-(6SO3-GlcNAc)-[D] (II) wherein (6SO3-GlcNAc) represents a 6-sulfated N-acetylglucosamine residue, (GlcNAc) represents an N-acetylglucosamine residue, and [D] represents a hydroxyl group, a substituted hydroxyl group or a saccharide residue, characterized in that a sulfated saccharide represented by the following general formula (IIa): HO-(6SO3-GlcNAc)-[D] (IIa) wherein (6SO3-GlcNAc) and [D] have the same meaning as above, is reacted with a saccharide of the following general formula (IIb): HO-[GlcNAc]-[Z] (IIb) wherein [GlcNAc] represents an N-acetylglucosamine residue and [Z] represents a hydroxyl group, a substituted hydroxyl group or a saccharide residue, in the presence of N-acetylhexosaminidase or N-acetylglucosaminidase. |
<SOH> BACKGROUND ART <EOH>As saccharides and glycolipids having a sulfate group in their molecules, sulfated sialyl-Lewis X, sulfatides, etc. are known. They are also known to specifically bind with selectin proteins which are present in human and animal leukocytes. Thus, the sulfated saccharides which show selectin blockers are expected to be developed and utilized as medicines such as anti-inflammatory medicines and cancer metastasis-preventing medicines. Various sulfated saccharides and methods for the preparation thereof have been hitherto developed. Such methods include as follows: (1) method of recovering from natural products by separation methods such as extraction and chromatography; (2) method using enzymes; (3) method by chemical synthesis; and (4) method combining the above methods (2) and (3). With the method (1) above, the desired product is generally obtained in only a trace amount. While the method (3) above can give the desired product in a large amount, much labors are required because the method includes many reaction steps. Concerning the method (2) above, the are relatively a large number of reports pertaining to the synthesis of sulfate group-free saccharides. Only a few synthesis examples are known with regard to sulfate group-containing saccharides. Incidentally, known methods of synthesizing sulfated saccharides using enzymes may be classified into two groups depending upon the kind of enzymes used. (a) Synthesis using sulfotransferase GlcNAcβ1→4GlcNAc→GlcNAcβ1→4 ( 6 SO 3 ) GlcNAc GlcNAcβ1→4GlcNAcβ1→4GlcNAcβ1→4GlcNAc→GlcNAcβ1→4GlcNAcβ1→4GlcNAcβ1→4 (6SO 3 ) GlcNAc The above synthesis method is disclosed in C.-H. Wong, J. Org. Chem., 65, 5565-5574 (2000); J. Am. Chem. Soc., 117, 8031 (1955). (b) Synthesis using galactotransferase 6SO 3 -GlcNAcβ1→3Galβ1→4Glcβ→OMPM→Galβ1→44 (6SO 3 ) GlcNAcβ1→3Galβ→OMPM The above synthesis method is disclosed in Lubineau, Carbohydrate Res., 305, 510 (1998). The reactions using the above enzymes are indicated as being useful as reactions per se but encounter great difficulties in actually carrying out the reactions. The method (a) is characterized in the use of a transferase enzyme (sulfotransferase) capable of transferring a sulfate group. The transferase enzyme is, however, expensive so that the sulfated saccharide product produced by using the expensive enzyme is unavoidably expensive too. Thus, such a method cannot be utilized for actual production on an industrial scale because of the production costs. Further, the position to which a sulfate group is transferred is limited to a 6 position on the reducing terminal side, i.e. it is difficult to synthesize sulfated saccharides in which the sulfate group is introduced to a non-reducing terminal. Therefore, the method (a) cannot be said as being a practical method. In the method (b), a transferase enzyme (galactotransferase) capable of transferring a sulfate group is used. The enzyme per se is currently used in various reactions and is in an already developed state. This enzyme, too, is expensive. Further, the substrate utilizable for this method is considerably limited. Therefore, the method (b) cannot be said to be a practical method. The objects of the present invention are as follows: (1) to provide a novel sulfated saccharide having a structure in which a sulfated saccharide chain is introduced into a non-reducing terminal of a saccharide, and to provide a method of producing the sulfated saccharide; and (2) to provide a novel sulfated saccharide having a structure in which a sulfated saccharide chain is introduced into a reducing terminal of a saccharide, and to provide a method of producing the sulfated saccharide. detailed-description description="Detailed Description" end="lead"? |
Use of dicatonic compounds for dyeing human keratinous fibres and compositions containing the same |
The present disclosure relates to the process of dyeing human keratinous fibers, such as the hair, with at least one specific direct dye in cosmetic compositions. Also disclosed herein are the direct dyeing and lightening compositions comprising said compounds, and the corresponding dyeing kits. |
1-18. (Canceled). 19. A process for dyeing human keratin fibers, comprising applying to the keratin fibers a cosmetic dye composition comprising at least one cationic direct dye compound chosen from those of formulae (I), (II) and (III): wherein: A and A1, which may be identical or different, are chosen from radicals of formula (a) Z is chosen from aliphatic and aromatic radicals, Z1 is an alkyl radical, R1 and R2, which may be identical or different, are chosen from hydrogen atoms, (C1-C4)alkyl radicals optionally substituted with at least one entity chosen from halogen atoms, hydroxyl radicals, carboxyl radicals, and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, or R1 and R2 can form, together with the two nitrogen atoms that bear them and the radical Z, a piperazine ring, X is a bridging radical chosen from —CO—; —CO—CH2—CH2—CO—; —CO—CO—; 1,4-dicarbonylphenyl; —CH2—CH2—; and triazines of formulae (b) and (c): wherein: Y and Y1, which may be identical or different, are chosen from halogen atoms and hydroxyl, amino, monoalkylamino, dialkylamino, 1-piperidino, morpholino and 1-piperazino radicals, wherein the piperazino radicals are optionally substituted on the nitrogen atom not attached to the triazine ring with at least one (C1-C4)alkyl radical, said alkyl radicals being optionally substituted with at least one radical chosen from hydroxyl, amino, mono(C1-C4)alkylamino and di(C1-C4)alkylamino radicals, Z2 is an (C2-C8)alkylene radical or forms a piperazine ring with the two adjacent nitrogen atoms and the radicals R1 and R2, R3 and R4, which may be identical or different, are chosen from hydrogen atoms, (C1-C4)alkyl radicals optionally substituted with at least one entity chosen from halogen atoms, hydroxyl, carboxyl, and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, R5 and R6, which may be identical or different, are chosen from hydrogen atoms; (C1-C4)alkyl and (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl, carboxyl, halogen and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, An− is an anion; X′ and Y′, which may be identical or different, are chosen from hydrogen atoms, halogen atoms, and (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkylcarbonylamino, arylcarbonylamino, ureido and arylureido radicals, R′1, is chosen from hydrogen, optionally substituted alkyl and aryl radicals, and R′2 radicals; R′2 is a radical of formula (d): wherein: B is chosen from linear and branched alkylene radicals, R′6 is chosen from hydrogen atoms and optionally substituted alkyls, R′7 and R′8, which may be identical or different, are chosen from optionally substituted alkyl radicals, R′6 and R′7, together with the nitrogen, form an optionally substituted 5-, 6- or 7-membered ring, which may comprise other hetero atoms, or alternatively R′6, R′7 and R′8 together form a pyridinium ring, R′3 is chosen from hydrogen atoms, halogen atoms, and (C1-C4)alkyl and (C1-C4)-alkoxy radicals, W is a radical of formula (e): wherein: K is a coupling radical, Z is a bridging radical chosen from the radicals of formulae: wherein R′9 is chosen from hydrogen atoms and optionally substituted (C2-C4)alkyl radicals, and B1 is chosen from linear and branched C2-C12 alkylene radicals optionally interrupted with at least one entity chosen from —NR′9— radicals, oxygen atoms and sulfur atoms; and wherein in formula (III), the number of cationic charges is two. 20. The process according to claim 19, wherein, in formula (I), R1 and R2, which may be identical or different, are chosen from hydrogen atoms, (C1-C4)alkyl radicals optionally substituted with a hydroxyl radical, and (C1-C4)alkoxy radicals, Z is chosen from linear, branched and cyclic C2-C8 alkyl radicals optionally substituted with an entity chosen from halogen atoms, hydroxyl and alkoxy radicals, the chain of said C2-C8 radicals optionally being interrupted with an entity chosen from oxygen atoms and —NR1— radicals; 1,4-phenyl radicals, 1,4-naphthyl radicals optionally substituted with at least one entity chosen from alkyl and alkoxy radicals and halogen atoms; and wherein Z may form a piperazine ring with R1, R2 and the two nitrogen atoms, and X is a group of formula (b). 21. The process according to claim 20, wherein, in formula (I), R1 and R2, which may be identical or different, are chosen from hydrogen atoms and methyl radicals. 22. The process according to claim 21, wherein, in formula (I), Z is chosen from unsubstituted phenyl radicals; phenyl and naphthyl radicals substituted with at least one radical chosen from methyl and methoxy radicals; (C2-C4)alkylene radicals optionally substituted with at least one hydroxyl radical; and a piperazine radical formed by bonding with R1, R2 and the two nitrogen atoms. 23. The process according to claim 19, wherein, in formula (II), Z1 is chosen from linear, branched and cyclic C2-C8 alkyl radicals optionally substituted with an entity chosen from halogen atoms, hydroxyl and alkoxy radicals, the chain of said C2-C8 alkyl radicals optionally being interrupted by an entity chosen from oxygen atoms and —NR1— radicals; and a piperazine ring formed with R1, R2 and the two nitrogen atoms, R3 and R4 are chosen from methyl and ethyl radicals, and R5 and R6 are chosen from hydrogen atoms, and methyl and methoxy radicals. 24. The process according to claim 23, wherein, in formula (II), Z1 is chosen from (C2-C6)alkylene radicals which are optionally substituted with at least one hydroxyl radical, and a piperazine ring formed with R1, R2 and the two nitrogen atoms. 25. The process according to claim 24, wherein, in formula (II), Z1 is chosen from unsubstituted (C2-C4)alkylene radicals. 26. The process according to claim 19, wherein, in formula (III), W is a radical of formula (e), wherein K is a coupling compound chosen from those of formula (f), (g) and (h): wherein, X′ and Y′, which may be identical or different, are chosen from hydrogen atoms, halogen atoms, and (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkylcarbonylamino, arylcarbonylamino, ureido and arylureido radicals, R′1 is chosen from hydrogen, optionally substituted alkyl and aryl radicals, and R′2 radicals; R′2 is a radical of formula (d): wherein: B is chosen from ethylene, n-propylene, isopropylene and n-butylene, R′6 is chosen from hydrogen atoms and optionally substituted alkyls, R′7 and R′8, which may be identical or different, are chosen from optionally substituted alkyl radicals, R′6 and R′7, together with the nitrogen, form an optionally substituted 5-, 6- or 7-membered ring, which may comprise other hetero atoms, or alternatively R′6, R′7 and R′8 together form a pyridinium ring, n is equal to 1 or 2, K1 is a radical of formula: 27. The process according to claim 19, wherein the at least one compound of formula (I) is chosen from those of formulae (I)1 to (I)7: 28. The process according to claim 19, wherein the at least one compound of formula (III) is the following compound: 29. A cosmetic composition for dyeing human keratin fibers comprising, in a physiologically acceptable medium, an effective amount of at least one direct dye chosen from those of formulae (I), (II) and (III): wherein: A and A1, which may be identical or different, are chosen from radicals of formula (a) Z is chosen from aliphatic and aromatic radicals, Z1 is an alkyl radical, R1 and R2, which may be identical or different, are chosen from hydrogen atoms; (C1-C4)alkyl radicals optionally substituted with at least one entity chosen from halogen atoms, hydroxyl, carboxyl and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, or R1 and R2 can form, together with the two nitrogen atoms that bear them and the radical Z, a piperazine ring, X is a bridging radical chosen from —CO—; —CO—CH2—CH2—CO—; —CO—CO—; 1,4-dicarbonylphenyl; —CH2—CH2—; and triazines of formulae (b) and (c): wherein: Y and Y1, which may be identical or different, are chosen from halogen atoms and hydroxyl, amino, monoalkylamino, dialkylamino, 1-piperidino, morpholino and 1-piperazino radicals, wherein the piperazino radicals are optionally substituted on the nitrogen atom not attached to the triazine ring with at least one (C1-C4)alkyl radical, said alkyl radicals being optionally substituted with at least one chosen from hydroxyl, amino, mono(C1-C4)alkylamino and di(C1-C4)alkylamino radicals, Z2 is an (C2-C8)alkylene radical or forms a piperazine ring with the two adjacent nitrogen atoms and the radicals R1 and R2, R3 and R4, which may be identical or different, are chosen from hydrogen atoms; (C1-C4)alkyl radicals optionally substituted with at least one entity chosen from halogen atoms, hydroxyl, carboxyl, and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, R5 and R6, which may be identical or different, are chosen from hydrogen atoms; (C1-C4)alkyl and (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl, carboxyl, halogen and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with a radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, An− is an anion; X′ and Y′, which may be identical or different, are chosen from hydrogen atoms, halogen atoms, and (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkylcarbonylamino, arylcarbonylamino, ureido and arylureido radicals, R′1, is chosen from hydrogen, optionally substituted alkyl and aryl radicals, and R′2 radicals; R′2 is a radical of formula (d): wherein: B is chosen from linear and branched alkylene radicals, R′6 is chosen from hydrogen atoms and optionally substituted alkyl radicals, R′7 and R′8, which may be identical or different, are chosen from optionally substituted alkyl radicals, R′6 and R′7, together with the nitrogen, form an optionally substituted 5-, 6- or 7-membered ring, which may comprise other hetero atoms, or alternatively R′6, R′7 and R′8 together form a pyridinium ring, R′3 is chosen from hydrogen atoms, halogen atoms, and (C1-C4)alkyl and (C1-C4)alkoxy radicals, W is a radical of formula (e): wherein: K is a coupling radical, Z is a bridging radical chosen from the radicals of formulae: wherein R′9 is chosen from hydrogen atoms and optionally substituted (C2-C4)alkyl radicals, and B1 is chosen from linear and branched C2-C12 alkylene radicals optionally interrupted with at least one entity chosen from —NR′9— radicals, oxygen atoms and sulfur atoms; and wherein in formula (III), the number of cationic charges is two. 30. The cosmetic composition according to claim 29, wherein the at least one direct dye chosen from those of formulae (I), (II) and (III) is present in an amount ranging from 0.01% to 40% by weight, relative to the total weight of the composition. 31. The cosmetic composition according to claim 30, wherein the at least one direct dye is present in an amount ranging from 0.1% to 20% by weight, relative to the total weight of the composition. 32. The cosmetic composition according to claim 29 further comprising at least one oxidizing agent. 33. The cosmetic composition according to claim 32, wherein the at least one oxidizing agent is chosen from hydrogen peroxide, urea peroxide, alkali metal bromates and ferricyanides, persalts, and redox enzymes, optionally with the respective donor or cofactor thereof. 34. The cosmetic composition according to claim 29, further comprising, in an amount ranging from 0.01% to 10% by weight, relative to the total weight of the composition, at least one cationic or amphoteric polymer chosen from: dimethyldiallylammonium chloride homopolymers; polymers comprising repeating units of formulae (W) or (U): polymers comprising units of formula (IX): wherein p is equal to 3, D is chosen from a bond, —(CH2)4—CO— groups, and —(CH2)7—CO— groups, X is a chlorine atom, or alternatively D may be chosen from block copolymers formed from units corresponding to the polymers when D is chosen from a bond and —(CH2)4—CO— groups; copolymers of acrylic acid and of dimethyldiallylammonium chloride. 35. The cosmetic composition according to claim 29, wherein, in formula (I), R1 and R2, which may be identical or different, are chosen from hydrogen atoms and (C1-C4)alkyl radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals, Z is chosen from linear, branched and cyclic C2-C8 alkyl radicals optionally substituted with at least one entity chosen from halogen atoms, hydroxyl and alkoxy radicals, the chain of said C2-C8 radicals optionally being interrupted with at least one entity chosen from oxygen atoms and —NR1— radicals; 1,4-phenyl radicals, 1,4-naphthyl radicals optionally substituted with at least one entity chosen from alkyl and alkoxy radicals and halogen atoms; and a piperazine ring formed with R1, R2 and the two nitrogen atoms, and X is a group of formula (b). 36. The cosmetic composition according to claim 35, wherein, in formula (I), R1 and R2, which may be identical or different, are chosen from hydrogen atoms and methyl radicals. 37. The cosmetic composition according to claim 36, wherein, in formula (I), Z is chosen from unsubstituted phenyl radicals, phenyl and naphthyl radicals substituted with at least one radical chosen from methyl and methoxy radicals, (C2-C4)alkylene radicals optionally substituted with at least one hydroxyl, and a piperazine radical formed by bonding with R1, R2 and the two nitrogen atoms. 38. The cosmetic composition according to claim 29, wherein, in formula (II), Z1 is chosen from linear, branched and cyclic C2-C8 alkyl radicals optionally substituted with an entity chosen from halogen atoms, hydroxyl and alkoxy radicals, the chain of said C2-C8 alkyl radicals optionally being interrupted by an entity chosen from oxygen atoms and —NR1— radicals; and a piperazine ring formed with R1, R2 and the two nitrogen atoms, R3 and R4 are chosen from methyl and ethyl radicals, and R5 and R6 are chosen from hydrogen atoms, and methyl and methoxy radicals. 39. The cosmetic composition according to claim 38, wherein, in formula (II), Z1 is chosen from (C2-C6)alkylene radicals which are optionally substituted with at least one hydroxyl radical, and a piperazine ring formed with R1, R2 and the two nitrogen atoms. 40. The cosmetic composition according to claim 39, wherein, in formula (II), Z1 is chosen from unsubstituted (C2-C4)alkylene radicals. 41. The cosmetic composition according to claim 29, wherein, in formula (III), B is chosen from ethylene, n-propylene, isopropylene and n-butylene, K is a coupling compound chosen from those of formula (f), (g) and (h): wherein, X′ and Y′, which may be identical or different, are chosen from hydrogen atoms, halogen atoms, and (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkylcarbonylamino, arylcarbonylamino, ureido and arylureido radicals, R′1 is chosen from hydrogen, optionally substituted alkyl and aryl radicals, and R′2 radicals; R′2 is a radical of formula (d): wherein: B is chosen from ethylene, n-propylene, isopropylene and n-butylene, R′6 is chosen from hydrogen atoms and optionally substituted alkyls, R′7 and R′8, which may be identical or different, are chosen from optionally substituted alkyl radicals, R′6 and R′7, together with the nitrogen, form an optionally substituted 5-, 6- or 7-membered ring, which may comprise other hetero atoms, or alternatively R′6, R′7 and R′8 together form a pyridinium ring, n is equal to 1 or 2, K1 is the radical of formula: 42. The cosmetic composition according to claim 29, wherein the at least one compound of formula (I) is chosen from those of formulae (I)1 to (I)7: 43. The cosmetic composition according to claim 29, wherein the at least one compound of formula (III) is the following compound: 44. The process according to claim 19, comprising applying the cosmetic dye composition onto wet or dry fibers, leaving it to act for a leave-in time ranging from 5 seconds to 60 minutes, rinsing the fibers and optionally washing them with shampoo, followed by rinsing them again and drying them. 45. The process according to claim 44, wherein the cosmetic dye composition further comprises at least one oxidizing agent. 46. The process according to claim 45, wherein when the cosmetic dye composition comprises at least one oxidizing agent, the at least one oxidizing agent is mixed with the cosmetic dye composition just before applying it to the fibers. 47. A multi-compartment dyeing kit, comprising at least one first compartment comprises a composition comprising at least one direct dye chosen from formulae (I), (II) and (III): wherein: A and A1, which may be identical or different, are chosen from radicals of formula (a) Z is chosen from aliphatic and aromatic radicals, Z1 is an alkyl radical, R1 and R2, which may be identical or different, are chosen from hydrogen atoms, (C1-C4)alkyl radicals optionally substituted with at least one entity chosen from halogen atoms, hydroxyl, carboxyl and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, or R1 and R2 can form, together with the two nitrogen atoms that bear them and the radical Z, a piperazine ring, X is a bridging radical chosen from —CO—; —CO—CH2—CH2—CO—; —CO—CO—; 1,4-dicarbonylphenyl; —CH2—CH2—; and triazines of formulae (b) and (c): wherein: Y and Y1, which may be identical or different, are chosen from halogen atoms and hydroxyl, amino, monoalkylamino, dialkylamino, 1-piperidino, morpholino and 1-piperazino radicals, wherein the piperazino radicals are optionally substituted on the nitrogen atom not attached to the triazine ring with at least one (C1-C4)alkyl radical, said alkyl radicals being optionally substituted with at least one radical chosen from hydroxyl, amino, mono(C1-C4)alkylamino and di(C1-C4)alkylamino radicals, Z2 is an (C2-C8)alkylene radical or forms a piperazine ring with the two adjacent nitrogen atoms and the radicals R1 and R2, R3 and R4, which may be identical or different, are chosen from hydrogen atoms, (C1-C4)alkyl radicals optionally substituted with at least one entity chosen from halogen atoms, hydroxyl, carboxyl, and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, R5 and R6, which may be identical or different, are chosen from hydrogen atoms, and (C1-C4)alkyl and (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl, carboxyl, halogen and cyano radicals; (C1-C4)alkoxy radicals optionally substituted with at least one radical chosen from hydroxyl and (C1-C4)alkoxy radicals; and amino, alkylamino, dialkylamino, aminocarbonyl, phenyl, phenoxy and phenylaminocarbonyl radicals, wherein the phenyl radicals are optionally substituted with at least one radical chosen from (C1-C4)alkyl, (C1-C4)alkoxy and phenoxy radicals, An− is an anion; X′ and Y′, which may be identical or different, are chosen from hydrogen atoms, halogen atoms, and (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkylcarbonylamino, arylcarbonylamino, ureido and arylureido radicals, R′1 is chosen from hydrogen, optionally substituted alkyl and aryl radicals, and R′2 radicals; R′2 is a radical of formula (d): wherein: B is chosen from linear and branched alkylene radicals, R′6 is chosen from hydrogen atoms and optionally substituted alkyls, R′7 and R′8, which may be identical or different, are chosen from optionally substituted alkyl radicals, R′6 and R′7, together with the nitrogen, form an optionally substituted 5-, 6- or 7-membered ring, which may comprise other hetero atoms, or alternatively R′6, R′7 and R′8 together form a pyridinium ring, R′3 is chosen from hydrogen atoms, halogen atoms, and (C1-C4)alkyl and (C1-C4)alkoxy radicals, W is a radical of formula (e): wherein: K is a coupling radical, Z is a bridging radical chosen from the radicals of formulae: and —CO—NR′9—B1—NR′9—CO—, wherein R′9 is chosen from hydrogen atoms and optionally substituted (C2-C4)alkyl radicals, and B1 is chosen from C2-C12 linear and branched alkylene radicals optionally interrupted with at least one entity chosen from —NR′9— radicals, oxygen atoms, and sulfur atoms; and wherein in formula (III), the number of cationic charges is two; and at least one second compartment comprising an oxidizing composition. |
Financial funding system and methods |
A financial funding system facilitates internet funding by a parent of a plurality of financial accounts that are set up for a student at a university. Preferably, the accounts correspond to expense items at the university such as tuition, bookstore, meals, and the like. A client presentation layer communicates with the parent and student, a security layer authenticates users, a business logic layer manages the accounts, a data layer stores information about the parent, student, the accounts and records transfers. An internet payment engine transfers funds. Methods include receiving communications from the parent or student, authenticating registered users, providing access to the plurality of accounts by the parent to fund same and by the student to pay corresponding expense items of the university, transferring funds such as with an internet payment engine, maintaining and updating the accounts, issuing email notification of the transfer of funds, and funding of multiple students at multiple universities. |
1. A financial funding system to facilitate funding by a parent of a plurality of financial accounts set up for a student, wherein said parent communicates with said financial funding system via the internet, said system comprising: a client presentation layer to communicate via the internet with said parent; a security layer to provide authentication of said parent; a business logic layer to manage a plurality of financial accounts set up for said student and to enable said parent to transfer funds from a parent's account to selected ones of said plurality of financial accounts for the student; an internet payment engine for electronically transferring funds from the parent's account to selected ones of said financial accounts of said student; and a data layer for storing information about the parent, the student, the plurality of financial accounts and for keeping records of any fund transfers. 2. The financial funding system in accordance with claim 1 wherein at least some of said plurality of financial accounts are set up to fund specific expense items of a university. 3. The financial funding system in accordance with claim 2 wherein said student may communicate over the internet with said client presentation layer and may use said business logic layer to authorize payments from selected ones of said plurality of financial accounts to pay certain expense items of the university. 4. The financial funding system in accordance with claim 3 wherein said expense items may be selected from a group consisting of meals, tuition, bookstore and/or activities. 5. The financial funding system in accordance with claim 1 further comprising: a notification layer for issuing electronic mail notification of any transfer of funds to a group consisting of the parent and/or the student. 6. The financial funding system in accordance with claim 1 further comprising: a financial institution for maintaining and updating said plurality of financial accounts. 7. The financial funding system in accordance with claim 1 wherein said parent may fund a plurality of financial accounts set up for a plurality of students with at least some of said plurality of students enrolled at different universities. 8. A method of facilitating a parent to fund a plurality of financial accounts in a financial funding system, said parent in communication with said financial funding system via the internet, said method comprising the steps of: receiving communications from said parent via the internet; authenticating said parent as a registered user of the financial funding system; providing access to the plurality of financial accounts for a student associated with the parent; receiving authorization for amounts to be transferred into selected ones of said plurality of financial accounts of said student; and transferring funds from an account of the parent to selected ones of said plurality of financial accounts of said student. 9. The method as claimed in accordance with claim 8 comprising the additional step of: storing information concerning said parent, said student, said plurality of accounts in a database of the financial funding system. 10. The method as claimed in accordance with claim 8 comprising the additional step of: keeping records of any fund transfers in a database of the financial funding system. 11. The method as claimed in accordance with claim 8 comprising the additional step of: setting up said plurality of financial accounts to fund specific expense items of a university. 12. The method as claimed in accordance with claim 11 comprising the additional steps of: receiving communications from said student via the internet; authenticating said student as a registered user of the financial funding system; providing access to the plurality of financial accounts of said student; receiving authorization for amounts to be transferred out of selected ones of said plurality of financial accounts of said student; and transferring funds out of selected ones of said plurality of financial accounts of said student to pay for corresponding expense items of the university. 13. The method as claimed in accordance with claim 8 comprising the additional step of: issuing an electronic mail notification of the transfer of funds to a group consisting of the parent and/or the student. 14. The method as claimed in accordance with claim 8 comprising the additional step of: maintaining and updating the plurality of financial accounts of said student at a financial institution. 15. The method as claimed in accordance with claim 8 comprising the additional step of: funding a plurality of financial accounts for a second student wherein said second student is at a different university than said student. 16. The method as claimed in accordance with claim 8 comprising the additional step of: using an internet payment engine to transfer funds from an account of said parent to selected ones of said plurality of financial accounts of said student. 17. The method as claimed in accordance with claim 12 comprising the additional step of: using an internet payment engine to transfer funds from selected ones of said plurality of financial accounts of said student to pay for corresponding expense items of said university. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates generally to online fund transfer and payment services and, more particularly, to systems and methods that utilize an internet payment engine and corresponding portal to enable funders to have greater flexibility and control over the funding of financial accounts and the payment of financial obligations. Provision of financial services to students at universities affects three core constituencies: 1. the university, including its staff, 2. the students and 3. the parents, relatives, guardians or others (hereinafter “parents” or “funder”) that are providing part or all of the funding for any student's education. Each constituency has unique, but complementary needs. For example, university administrators face the most complex challenges of these three constituencies due to their need to facilitate funds transfers from parents to students and manage payment processing for core, non-academic services. They further need to manage and reconcile these fund transfers and receivables accounts. Still further, they need to facilitate access to on campus financial services for their students. The universities are presented with payment processing challenges due to acceptance of funds into and tracking university “declining balance” and/or credit programs. These consume significant time and administrative resources throughout the university. A variety of university departments have separate processes and systems to meet similar account funding needs including, among others, tuition, boarding, food services, bookstores, athletic activities, financial services, and the like. The universities are also presented with account management challenges. Among others, the university must manage the paper trail left behind by checks and credit card payments made on behalf of thousands of students throughout the year. The universities must further integrate and balance deferring programs and accounts. Still further, the universities must reconcile multiple independent accounts, payment systems and procedures. The universities also face on-campus financial services challenges. Although universities are not generally in the banking business, university personnel often wish to assist students with accessing financial services. In particular, students arriving on campus for the first time often have little financial experience. Most do not have a bank account or ready access to cash or depository services. In view of the foregoing, universities and colleges need assistance in organizing, funding and managing the range of campus programs funded by parents and students throughout the year. Universities can benefit from all components of the system of the present invention. Students, the second core university constituency, typically do not have established banking patterns or relationships. Most current students are highly active in electronic commerce and/or the internet. While many current students do not have experience with financial services, such students would be more likely to accept widespread automated teller machine card and credit card usage. Current students who are in need of a banking account and/or relationship, are less likely to select their bank on location and are likely to utilize internet banking, including direct deposits and withdrawals. Parents face several challenges in providing for the day-to-day financial needs of college students. Many students are away from home for the first time, but will remain financially dependent on their parents. As a result, parents need to quickly and easily provide spending money, allowances, pay for tuition, pay for books and supplies, fund meal plans and various other accounts. Often, universities may define sub-accounts for the various expenses that may be encountered or anticipated. Many parents also wish to know that funds provided for such specific needs are actually used for their intended purpose, and that the necessary payments have, in fact, been made. It is a general object of the present invention to provide systems and methods that utilize an internet payment engine and a corresponding portal to enable funders to have greater flexibility and control over the funding of financial accounts and the payment of financial obligations, such as in a university environment. Another object of the present invention is to provide a system and methods of online funding for the financial needs of university students. Yet another object of the present invention is to provide a system and methods to reduce the time and resources required by a university to manage, process and track the financial transactions of the university students. A further object of the present invention is to provide a system and methods of providing financial services to university students via electronic commerce and the internet. A still further object of the present invention is to provide a system and methods for parents to conveniently fund the financial needs of students and to track financial transactions so that the parents can determine that the funds were used for the intended purposes. Another object of the present invention is to provide a system and methods to enable parents to conveniently fund the financial needs of multiple students that may be enrolled in different universities. |
<SOH> SUMMARY OF THE INVENTION <EOH>The systems and methods of the present invention are concerned with providing unique financial services for university communities, and are particularly applicable to financial accounts for university students that are directly or indirectly set up by the university. These systems and methods provide online funding of various expense items associated with a university, such as meal plans, bookstores, tuition, activities, personal expenses and other university programs. Preferably, a plurality of financial accounts are set up, one for each anticipated expense item. Parents can fund the accounts via the internet and students can issue payments from the accounts via the internet. These systems and methods thus provide financial services to three core university constituencies, including university administration and staff, the students and the parents. The financial funding system includes a client presentation layer that communicates with the parents and the students and that provides appropriate screens for each step of the funding or payment processes. A security layer authenticates the parents and students as registered users of the system. A business logic layer manages the accounts and permits parents to add funds to selected accounts and permits students to make payments from the funded accounts. An internet payment engine electronically transfers funds into the accounts and transfers payments out of the accounts. A data layer stores information about the registered parents and registered students, keeps records of the accounts and any transfers of funds to or from the accounts, and may also store preference and configuration information. A notification layer may issue emails concerning any transfer of funds, such as to the parents and/or students. The methods include receiving internet communications from the parents and students, authenticating the parents and students as authorized users of the system, providing access to a plurality of financial accounts for each student, receiving authorization for amounts to be transferred into or out of selected ones of the plurality of financial accounts, and transferring funds into or out of the selected accounts. The methods also include storing information about registered parents and students, keeping records of any fund transfers into or out of the accounts, setting up the accounts to fund corresponding expense items of the university, issuing email confirmations of any transactions to the parents and/or the students, maintaining and updating the financial accounts and using an internet payment engine to transfer the funds to or from the accounts. Yet another method concerns funding of multiple students enrolled at different universities. |
Novel compositions |
The present invention relates to compositions comprising DNA attached to one or more functional moities via a locked nucleic acid oligonucleotide. In particular the present invention provides compositions comprising a plasmid containing a gene encoding a protein of interest, wherein said plasmid may be introduced to a tissue or cell and the gene expressed, complexed to the locked nucleic acid functional moiety |
1. A locked nucleic acid conjugate comprising an oligonucleotide comprising at least one locked nucleic acid base and a functional moiety. 2. A locked nucleic acid conjugate as claimed in claim 1, wherein the functional moiety is a member selected from the group consisting of: Fluorescent labels; nuclear localisation peptides; peptides that have the ability to cross the plasma membrane of eukaryotic cells (“cell penetrating peptides”); endosomal escape peptides; cell targeting and binding peptides or protein; peptides or proteins with transcription activation domains; and molecules having adjuvant or immunostimulatory activity. 3. A locked nucleic acid conjugate as claimed in claim 1 wherein the oligonucleotide is between 7 to 25 bases in length. 4. A locked nucleic acid conjugate as claimed in claim 1 wherein at least 50% of the bases are locked nucleic acid bases. 5. A locked nucleic acid conjugate as claimed in claim 1 wherein the at least one locked nucleic acid base is a member selected from the group consisting of O—, (oxy) S—, (thio) ad NH—2 (amino) bridged locked nucleic acid base. 6. A locked nucleic acid conjugate as claimed in claim 1 wherein the oligonucleotide is free from self-complementary base pairings. 7. A locked nucleic acid conjugate as claimed in claim 1 comprising a cleavable linkage between the functional moiety and the oligonucleotide which is selectively cleavable after administration to a patient. 8. A locked nucleic acid conjugate as claimed in claim 1 wherein the functional moiety is an immunostimulatory oligonucleotide containing at least one unmethylated CG di-nucleotide motif. 9. A complex comprising a locked nucleic acid conjugate as claimed in claim 1, and a DNA sequence having a complementary sequence to the oligonucleotide, and encoding a gene under the control of a promoter. 10. A complex as claimed in claim 9 wherein at least one further locked nucleic acid conjugate is present which is bound to a complementary sequence within the locked nucleic acid complex which is itself bound to the DNA sequence. 11. A complex as claimed in claim 10 comprising an array of locked nucleic acid conjugates bound to a single locked nucleic acid complementary sequence within the DNA, formed by locked nucleic acid: locked nucleic acid hybridisation between locked nucleic acid oligonucleotide. 12. A complex as claimed in claim 9 wherein the gene encodes for a therapeutic protein or an antigen. 13. A complex as claimed in claim 10 wherein a plurality of locked nucleic acid complexes are bound to a plurality of complementary sequences within said DNA sequence. 14. A complex of claim 12 wherein the antigen is capable of raising an immune response against a pathogen or a tumour. 15. A complex as claimed in claim 9 wherein the DNA sequence is in the form of an open circular or supercoiled plasmid. 16. A pharmaceutical composition comprising a complex as claimed in claim 9 and a pharmaceutically acceptable carrier or dilulent. 17. A pharmaceutical composition as claimed in claim 16 wherein the complex is coated on to a microprojectile. 18. A pharmaceutical composition as claimed in claim 17 wherein the microprojectiles are gold beads. 19. A device loaded with the pharmaceutical composition of claim 16. 20-23. cancelled. 24. A process for the the preparation of a pharmaceutical composition comprising the step of hybridising the locked nucleic acid conjugate of claim 1 with a plasmid capable of expressing a gene encoding an antigen or therapeutic protein, and formulating the resulting complex with a pharmaceutical acceptable carrier. 25. An oligonucleotide comprising a first region comprising an oligonucleotide sequence having at least one locked nucleic acid, and a second region comprising an immunostimulatory oligonucleotide region containing at least one unmethylated CG di-nucleotide motif. 26. An oligonucleotide as claimed in claim 25 wherein the first locked nucleic acid containing region and the second immunostimulatory oligonucleotide region are separated by a phosphoramidate region, and wherein the second immunostimulatory oligonucleotide region comprises a phosphorothioate backbone. |
Methods for inducing reversible stasis |
The present invention concerns compositions and methods involving incubating biological materials under hypoxic or anoxic conditions to induce stasis or suspended animation. Methods of screening for compounds that induce stasis or compounds that increase the ability to undergo stasis are included. Such methods have ramifications for preserving biological materials as well as reducing or preventing trauma to biological materials. Also contemplated are methods for screening compounds that are active or more active under hypoxic conditions than normoxic conditions. Such methods can be used to identify antitumor compounds that would operate under hypoxic conditions in which tumor cells survive. |
1. A method for cryopreserving biological material comprising: a) first incubating the biological material under anoxic conditions for an effective amount of time for the biological material to enter stasis; and b) then cryopreserving the biological material. 2. The method of claim 1, wherein the biological material is a cell, tissue, or organism. 3. The method of claim 2, wherein the biological material is a cell. 4. The method of claim 3, wherein the cell is a sex cell. 5. The method of claim 3, wherein the cell is comprised in an embryo. 6. The method of claim 5, wherein the embryo is a vertebrate embryo. 7. The method of claim 6, wherein the vertebrate embryo is mammalian. 8. The method of claim 2, wherein the biological material is tissue. 9. The method of claim 8, wherein the tissue is an organ. 10. The method of claim 2, wherein the biological material is an organism. 11. The method of claim 10, wherein the organism is an invertebrate. 12. The method of claim 10, wherein the organism is a vertebrate. 13. The method of claim 1, wherein the biological material is incubated under anoxic conditions for more than 30 minutes. 14. The method of claim 13, wherein the biological material is incubated under anoxic conditions for more than 1 hour. 15. The method of claim 13, wherein the biological material is incubated under anoxic conditions for more than 2 hours. 16. The method of claim 1, wherein cryopreserving the biological material comprises perfusing the biological material with a cryoprotectant and lowering the temperature of the biological material. 17. The method of claim 16, wherein the temperature is lowered to below 0° C. 18. A method for preserving biological material comprising: a) incubating the biological material under hypoxic conditions for an effective amount of time for the biological material to enter stasis. 19. The method of claim 18, further comprising lowering the temperature of the biological material. 20. The method of claim 18, further comprising incubating the biological material under normoxic conditions to reverse stasis. 21. The method of claim 18, wherein the biological material is incubated under conditions of less than 10% oxygen. 22. The method of claim 21, wherein the biological material is incubated under conditions of less than 5% oxygen. 23. The method of claim 18, wherein the biological material exhibits signs of trauma. 24. The method of claim 19, wherein the temperature is lowered to below 15° C. 25. The method of claim 24, wherein the temperature is lowered to below 10° C. 26. The method of claim 18, wherein the biological material is an organism. 27. The method of claim 18, wherein the organism is a vertebrate. 28. The method of claim 27, wherein the vertebrate is a Danio rerio embryo. 29. A method for screening for an antitumor compound comprising: a) incubating a first anoxia-resistant organism under hypoxic conditions sufficient to permit the organism to enter stasis; b) incubating the first organism with a candidate compound; c) observing the first organism for viability; and d) comparing the first organism's viability against a second anoxia-resistant organism's viability incubated under normoxic conditions in the presence of the candidate compound, wherein viability of the second organism and lack of viability of the first organism identifies the candidate compound as an anti-tumor compound. 30. The method of claim 29, wherein the hypoxic conditions have less than 10% oxygen. 31. The method of claim 30, wherein the hypoxic conditions are anoxic. 32. The method of claim 29, further comprising removing the candidate compound from the first and second organisms. 33. The method of claim 29, wherein observing the first organism for viability comprises observing them for movement. 34. The method of claim 29, wherein the first and second anoxia-resistant organisms are nematodes. 35. The method of claim 34, wherein the nematode is Caenorabditis elegans. 36. The method of claim 29, wherein the first and second anoxia-resistant organisms are vertebrate organisms. 37. The method of claim 36, wherein the vertebrate organisms are embryos. 38. The method of claim 37, wherein the embryos are Danio rerio. 39. The method of claim 29, wherein the organisms are in a hypoxic environment for at least 30 minutes. 40. The method of claim 39, wherein the organisms are in a hypoxic environment for at least 1 hour. 41. The method of claim 40, wherein the organisms are in a hypoxic environment for at least 2 hours. 42. An antitumor composition comprising an antitumor compound identified by a process comprising: a) incubating a first anoxia-resistant organism under hypoxic conditions sufficient to induce stasis; b) incubating the first organism with a candidate compound; and c) comparing the first organism's viability against a second anoxia-resistant organism's viability incubated under normoxic conditions in the presence of the candidate compound, wherein the compound is an antitumor compound if the first anoxia-resistant organism is no longer viable and the second anoxia-resistant organism is viable after incubation with the candidate compound. 43. The composition of claim 42, wherein the first and second anoxia-resistant organisms are nematodes. 44. The composition of claim 42, wherein the first and second anoxia-resistant organisms are embryos. 45. The composition of claim 44, wherein the first and second anoxia-resistant organisms are vertebrate embryos. 46. The composition of claim 42, wherein the hypoxic conditions are less than 10% oxygen. 47. The composition of claim 46, wherein the hypoxic conditions are anoxic. 48. The composition of claim 47, wherein the first organism is incubated under hypoxic conditions for more than 30 minutes. 49. The composition of claim 48, wherein the first organism is incubated under hypoxic conditions for more than 1 hour. 50. The composition of claim 42, wherein the candidate compound is a small molecule. 51. The composition of claim 42, wherein the anti-tumor compound comprises mitotracker red. 52. A method for killing cancer cells in a patient with a tumor comprising administering to the patient an therapeutically effective amount of an antitumor compound identified by the process comprising: a) incubating a first anoxia-resistant organism under hypoxic conditions sufficient to permit the organism to enter stasis; b) incubating the first organism with a candidate compound; c) observing the first organism for viability; and d) comparing the first organism's viability against a second anoxia-resistant organism's viability incubated under normoxic conditions in the presence of the candidate compound, wherein viability of the second organism and lack of viability of the first organism identifies the candidate compound as an anti-tumor compound. 53. The method of claim 52, wherein the compound is administered directly to the tumor. 54. The method of claim 53, wherein the compound is injected into the tumor. 55. The method of claim 52, further comprising administering to the patient chemotherapy, radiotherapy, immunotherapy, or gene therapy. 56. The method of claim 52, further comprising resecting at least a portion of the tumor from the patient. 57. The method of claim 52, wherein the compound is mitotracker red. 58. A method for screening for a compound that induces stasis in biological material comprising: a) incubating a first organism capable of stasis with a candidate compound; and b) evaluating the first organism for stasis, wherein the compound is a stasis inducer if the first organism exhibits stasis after exposure to the compound. 59. The method of claim 58, further comprising c) comparing the ability to enter stasis in the first organism with a second organism not incubated or no longer incubated with the candidate compound. 60. The method of claim 58, further comprising d) removing the compound from the first organism; and e) evaluating the first organism for loss of stasis, wherein the compound is a reversible stasis inducer if the first organism exhibits stasis after incubation with the compound, but no longer exhibits stasis after the compound is removed. 61. A method of screening for a compound that improves the ability to undergo stasis comprising: a) incubating a first organism capable of undergoing stasis under hypoxic conditions; b) exposing the first organism to a candidate compound; c) incubating a second organism capable of undergoing stasis under the same hypoxic conditions as the first organism; d) comparing the first organism and the second organism. 62. A stasis inducer compound identified by a process comprising: a) incubating an organism capable of entering stasis with a candidate compound; and b) evaluating the organism for stasis, wherein the compound is a stasis inducer if the organism exhibits stasis after incubation with the compound. 63. A reversible stasis inducer compound identified by a process comprising: a) incubating an organism capable of entering stasis with a candidate compound; b) comparing the organism with an organism capable of entering stasis that is no longer incubated with the candidate compound, wherein the compound is a stasis inducer if the organism exhibits stasis after incubation with the compound, but does not exhibit stasis when no longer incubated with the compound. 64. The reversible stasis inducer compound of claim 63, wherein the organism no longer exposed to the candidate compound in c) is the organism of a). 65. A method of inducing stasis in biological material comprising administering to the biological material a stasis inducer compound. 66. The method of claim 65, further comprising lowering the temperature of the biological material. 67. The method of claim 65, wherein the biological material is an organ. 68. The method of claim 65, wherein the biological material is tissue. 69. The method of claim 65, wherein the biological material is an organism. 70. The method of claim 69, wherein the organism is a vertebrate organism. 71. The method of claim 70, wherein the vertebrate organism is an embryo. 72. The method of claim 70, wherein the vertebrate organism is a mammal. 73. The method of claim 72, wherein the mammal is a human. 74. A method of inducing stasis in a cell of an organism comprising administering to the cell of the organism an effective amount of a stasis inducer compound identified by a process comprising: a) incubating an organism capable of entering stasis with a candidate compound; b) evaluating the organism for loss of stasis after the compound is removed, wherein the compound is a stasis inducer if the organism exhibits stasis after incubation with the compound, but no longer exhibits stasis after the compound is removed. 75. A method of inducing stasis in an organism comprising administering to the organism an effective amount of a stasis inducer compound identified by a process comprising: a) incubating an organism capable of entering stasis with a candidate compound; b) evaluating the organism for loss of stasis after the compound is removed, wherein the compound is a stasis inducer if the organism exhibits stasis after incubation with the compound, but no longer exhibits stasis after the compound is removed. 76. A method of identifying a modulator of stasis in a biological material undergoing stasis comprising: a) incubating a first biological material undergoing stasis with a candidate compound, wherein the first biological material is incubated under hypoxic conditions; b) evaluating the first biological material for loss of stasis; c) comparing the first biological material with a second biological material undergoing stasis but not incubated with the candidate compound, wherein a difference in stasis between the first and second biological materials identifies the compound as a candidate modulator of stasis. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates generally to the fields of cell biology and physiology, as well as oncology. More particularly, it concerns methods and compositions involving exposing cells, tissues, or organisms to hypoxic or anoxic conditions. Compounds and methods for preserving and preventing damage to biological materials are specifically contemplated. Also contemplated are methods for screening compounds for the ability to induce stasis, or suspended animation, as well as for compounds with antitumor activity, and therapeutic compositions thereof. 2. Description of Related Art Most animals are very sensitive to reduced levels of oxygen. Known vertebrate responses to low oxygen concentrations (hypoxia) include changes in carbohydrate metabolism, an increase in nitric oxide, and a stimulation of red blood cell and hemoglobin production (Guillemin et al, 1997). Hypoxia also can induce the expression of a select set of genes, including glycolytic enzymes, glycoprotein hormone erythropoeitin and the inducible nitric oxide synthatase (Guillemin et al., 1997; Iyer et al., 1998). Hypoxia inducing factor (HIF-1) has been shown to play a central role in this transcriptional response (Semenza, 1999[a]; Semenza, 1999[b]). Extreme hypoxia is central to the pathology of several diseases involving cardiac and pulmonary dysfunction (Semenza, 2000). Additionally, it is known that in certain solid tumors the cancerous cells that are hypoxic are more resistant to radiation and chemotherapy (Brown, 1999). Identification of the response organisms have to low oxygen tension may facilitate the development of treatment for rescue or prevention of damaged ischemic tissue, or for the destruction of tumor cells with low oxygen tensions. Given its central role in physiology, several animal model systems have been developed to understand the response organisms have to reduced oxygen levels. The ability to survive anoxia (0% O 2 ) has been observed in small invertebrate organisms that lack a circulatory system and are therefore able to rapidly adapt to changes in oxygen levels (Foe et al., 1985; Hochachka et al., 1993). It has been shown that some invertebrates, such as Caenorhabditis elegans, Artemia franciscana , and Drosophila melanogaster , have the ability to survive in the absence of molecular oxygen (anoxia) (Anderson, 1978; Van Voorhies et al., 2000; Hand, 1993; Foe et al., 1985). The brine shrimp A. franciscana has been shown to survive four years of continuous anoxia and its response includes an arrest of development, a decrease in intracellular pH, a reduction in protein synthesis, and an accumulation of heat shock proteins (Hand, 1993; Clegg, 1997). It has been shown that both C. elegans and D. melanogaster can survive at least one day of anoxia exposure by arresting development until oxygen supply is reestablished (Van Voorhies et al., 2000; Foe et al., 1985). The survival of anoxia likely depends on the organisms ability to curb energy usage by shutting down nonessential cellular functions, maintain stable and low permeability of membranes, and the ability to synthesize ATP by glycolytic processes (Hochachka, 1986; Hochachka et al., 1996). Recent studies in D. melanogaster and mammalian tissue have demonstrated that the nitric oxide/cyclic GMP signaling pathway is involved in the response to oxygen deprivation (Wingrove et al, 1999; Clementi et al., 1999; Giulivi, 1998). The ability to induce stasis (or suspended animation) in more developed organisms has not been previously demonstrated. This would provide ways of screening for stasis-inducing compounds that may have applicability to other vertebrate organisms, including mammals. Such applicability may extend to inducing stasis in cells, tissues, organs, systems, and entire organisms. Thus, the ability to suspend movement and/or development has ramifications with respect to short- or long-term preservation of biological material. In addition to the advantages of preservation by itself, preservation also may facilitate trauma or wound therapy, transportation of biological materials, as well as manipulation of biological materials. Furthermore, because anoxic and hypoxic conditions simulate conditions under which a tumor in an animal subsists, antitumor compounds can be identified using organisms susceptible to stasis. While antitumor (anticancer) therapies exist, there is a continued need for new or improved methods of treating tumors. The present invention demonstrates the ability to induce stasis in an organism and provides methods and compositions that address the needs identified above. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention takes advantage of the discovery that organisms, including vertebrate organisms can undergo stasis when incubated under anoxic or hypoxic conditions. The present invention comprises methods of inducing stasis in biological materials—including organisms—as well as methods of modulating biological materials undergoing stasis or in stasis. As discussed herein, the invention extends to biological materials including cells—fertilized and unfertilized—tissues, organs, and parts of organisms, and entire organisms. It is specifically contemplated that methods and compositions with respect to one type of biological material may be implemented with respect to all other types of biological materials. In many instances, the organism is a vertebrate, while in others it is an invertebrate. Where the organism is invertebrate, embodiments include, but are not limited to Caenorabditis elegans or C. elegans . Vertebrate organisms include mammals, reptiles, amphibians, birds, and fish. Mammals are specifically contemplated, including those of veterinary, agricultural, and research importance, such as canine, feline, bovine, ovine, porcine, caprine, rodent, lagomorph, and swine. Humans, are specifically contemplated to be organisms for which the methods of the invention are applicable. Fish, including those of veterinary and aquacultural importance include, but are not limited to, Danio rerio , salmon, catfish, halibut, tuna, sea bass, red snapper, dover sole, petrale sole, tilapia, swordfish, mahi mahi, mackerel, yellowtail, skipper jack, opa, amberjack, barracuda, black drum, black grouper, cobia, flounder, gag grouper, jack crevalle, jewfish, king mackerel, ladyfish, lane snapper, mangrove snapper, mutton snapper, permit, pompano, redfish, red grouper, sheepshead, snook, spanish mackerel, spotted seatrout, tarpon, tripletail, yellowtail snapper, other bony fish, as well as cartilaginous fish such as sharks and rays, and shellfish. Birds used in embodiments of the invention include, but are not limited to, chickens, geese, ducks, pheasants, ostriches, emu, quails, and turkeys. In some embodiments, stasis is induced in biological material by exposing or incubating the biological material under hypoxic or anoxic conditions sufficient to induce stasis of the biological material. It is contemplated that “sufficient to induce stasis” means that the material is exhibiting signs of stasis, i.e., for a finite length of time (as opposed to death) there is lack of movement, absence of cell division, reduction in cell division, absence of heartbeat, reduced heart beat, and lack of or reduction in developmental progression as observed by light microscopy. As discussed in further detail below, hypoxic conditions include conditions in which the oxygen concentration is less than 20.8%—the concentration of normal atmospheric conditions—and as low as 0% (anoxic conditions); thus hypoxic conditions includes anoxic conditions unless otherwise specified; it is contemplated that hypoxic conditions with more than 0% oxygen are part of the invention. In some embodiments, oxygen concentration is less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%. In further embodiments, oxygen concentration is 0% or greater, or is between 0.5% and 20.8%. Methods implemented under anoxic conditions may also be implemented under hypoxic conditions and vice versa; both are contemplated as part of the present invention. It is also contemplated that in most embodiments of the invention, biological material will be restored to normoxic conditions, allowing for stasis to be reversed. It is further contemplated that minimal damage or harm to the biological material will result from being in stasis under the conditions described herein. In all methods of the claimed invention, biological material may be exposed to temperatures lower than room temperature, including temperatures that will freeze the biological material, depending upon the liquid in or with which the biological material is incubated or perfused. Lowering of temperature may increase the duration that the biological material may undergo reversible stasis, preserve biological material, prevent damage or further damage to biological material, allow the biological material to undergo reversible stasis, increase the length of time biological material may be preserved, or increase the efficacy of a stasis inducer. In some embodiments, biological materials may be exposed to temperatures that allow the biological material to be frozen. For example, in some embodiments of the invention, sex cells or fertilized eggs are treated according to methods of the invention for use at a subsequent time. Alternatively, biological material may be incubated under hypoxic or anoxic conditions and placed in a temperature lower than room temperature either to prevent damage to biological material or to prevent further damage to biological material, such as to stave off the onset of trauma. Thus, in further embodiments, the present invention includes methods for cryopreserving biological material comprising: first incubating the biological material under hypoxic or anoxic conditions for an effective amount of time for the biological material to enter stasis; and then cryopreserving the biological material. Cryopreserving biological material may involve steps generally used in cryopreservation, including steps of vitrification. Therefore, in further embodiments of the invention, steps of perfusing biological materials, particularly organs or tissues, with cryoprotectant agents are contemplated as part of the invention to protect the biological material. In certain other embodiments of the present invention, methods are included for preserving biological material, particulalry organ or tissues. Such methods include first incubating the biological material under hypoxic or anoxic conditions for an effective amount of time for the biological material to enter stasis and then lowering the temperature of the biological material. In addition to hypoxic/anoxic conditions, another way of inducing stasis is to administer and effective amount of a stasis inducer compound, which is a compound capable of inducing a biological material to enter stasis, preferably reversible stasis. An “effective amount” of a compound, generally, refers to an amount sufficient to detectably and repeatedly achieve a particular result. In the context of the present invention, one result sought is to induce stasis or suspended animation in a biological material. An effective amount of a stasis inducer, for example, would eliminate any detectable movement of the biological material, including, if appropriate, any detectable movement in the whole organism. More rigorous definitions may apply, including reduction or inhibition of cellular metabolism. Alternatively, in some embodiments the particular result desired is the treatment of a cancer, particularly a tumor. A “therapeutically effective amount” refers to any amount of a substance that promotes or enhances the well-being of the patient with respect to the medical treatment of his cancer. A list of nonexhaustive examples of this includes extension of the patient's life by any period of time; decrease or delay in the neoplastic development of the disease; decrease in hyperproliferation; reduction in tumor growth; delay of metastases; reduction in the proliferation rate of a cancer cell or tumor cell; induction of apoptosis in any treated cell or in any cell affected by a treated cell; and a decrease in pain to the patient that can be attributed to the patient's condition. The present invention further concerns methods of screening for compounds that are candidates for cancer treatment. Such compounds may be antitumor compounds because of their ability to act under conditions of hypoxia, but not under conditions of normoxia. Alternatively, they may be stasis inducing compounds, that is, compounds that induce biological materials to undergo stasis. Furthermore, compounds that increase the efficacy of hypoxic conditions to induce stasis or that reduce any damage from stasis can be identified in screens of the present invention. The compounds to be screened include, but are not limited to small chemical molecules, peptides, polypeptides, nucleic acids, combinations and analogs thereof, which may be natural or synthetic products. Large-scale screening assays may be employed for screening methods of the invention. Libraries may be implemented, as well as high thoughput analysis. Methods of screening for an antitumor compound comprise: a) incubating a first anoxia- or hypoxia-resistant organism under hypoxic or anoxic conditions sufficient to permit the organism to enter stasis; b) incubating the first organism with a candidate compound; c) observing the first organism for viability; and d) comparing the first organism's viability against a second anoxia-resistant organism's viability incubated under normoxic conditions in the presence of the candidate compound. Viability of the second organism and lack of viability of the first organism identifies the candidate compound as an anti-tumor compound. It is specifically contemplated that any biological material may be implemented in this assay. “Anoxia-resistant” biological material (including anoxia-resistant organisms) have an ability to survive without oxygen without exhibiting harmful effects, which include, but are not limited to, developmental or physiological defects, such as brain damage, damage to the nervous system, or cardio-pulminary issues that result in tissue damage. “Hypoxia-resistant” biological material and organisms have an ability to survive under hypoxic conditions without exhibiting such harmful effects described above. The use of other biological materials, such as cells or tissues, is specifically contemplated for use with the present method of screening for antitumor compounds. In some embodiments, the candidate compounds are removed from the biological materials. Evaluating viability may include evaluating the biological materials for movement, cell division, developmental progression, or other metabolic activities. Evaluation of gross changes such as heartbeat, cell division, movement, and developmental progression may be evaluated using an optical aid, such as a light microscope and camera. Metabolic activities, such as phosphorylation or ATP:ADP ratios, can be evaluated by methodology well known to those of skill in the art. Compositions identified by the screening methods of the invention form part of the present invention. Thus, the invention includes an antitumor composition comprising an antitumor compound identified by a process comprising: a) incubating a first anoxia-resistant organism under hypoxic conditions sufficient to induce stasis; b) incubating the first organism with a candidate compound; and c) comparing the first organism's viability against a second anoxia-resistant organism's viability incubated under normoxic conditions in the presence of the candidate compound. As discussed earlier, in any embodiments of the invention, other biological material may be susbstituted for an organism. The compound is an antitumor compound if the first anoxia-resistant organism is no longer viable and the second anoxia-resistant organism is viable after incubation with the candidate compound. Any embodiment discussed above may be employed with this method and composition. A hypoxia-resistant organism may be employed with this method in place of an anoxic-resistant organism. Chloromethyl-X-rosamine (CAS registry number: 167095-09-2 1H, 5H, 11H, 15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[4-(chloromethyl)phenyl]-2,3,6,7,12,13,16,17-octahydro-, chloride; also known as (MITOTRACKER RED, Molecular Probes, Eugene Oreg.)) has been identified as compound that affects biological material under hypoxic conditions but not under normoxic conditions. This compound, or a derivative or analog thereof, may constitute an anti-cancer compound that can be employed as an anti-tumor compound for administration to a patient with a tumor. Also included as part of the invention are methods for killing tumor cells in a patient with a tumor comprising administering to the patient a therapeutically effective amount of an antitumor compound identified by the process comprising: a) incubating a first anoxia-resistant organism under hypoxic conditions sufficient to permit the organism to enter stasis; b) incubating the first organism with a candidate compound; c) observing the first organism for viability; and d) comparing the first organism's viability against a second anoxia-resistant organism's viability incubated under normoxic conditions in the presence of the candidate compound, wherein viability of the second organism and lack of viability of the first organism identifies the candidate compound as an anti-tumor compound. The method, in some embodiments, further comprises performing surgery on the patient or administering at least one other anti-cancer treatment, such as chemotherapy, radiotherapy, immunotherapy or gene therapy. The compound identified above as chloromethyl-X-rosamine (MITOTRACKER RED) is specifically contemplated for use in the present invention. Other screening methods include screening for a compound that induces stasis in biological material comprising: a) incubating a first biological material capable of undergoing stasis with a candidate compound; and b) evaluating the first biological material for evidence of stasis. The compound is a stasis inducer if the first biological material exhibits evidence of stasis after exposure to the compound. In some embodiments, the method further comprises c) comparing the ability to induce stasis in the first biological material with a second biological material not incubated or no longer incubated with the candidate compound. In still further embodiments, it also comprises d) removing the compound from the first biological material; and e) evaluating the first biological material for loss of stasis. The compound is a reversible stasis inducer if the first organism exhibits stasis after incubation with the compound, but no longer exhibits stasis after the compound is removed. In some embodiments, a reversible stasis inducer can be identified by comparing biological material that was exposed to the candidate compound and the same biological material but in the absence of the candidate compound or after the candidate compound has been removed. In still further embodiments, the present invention concerns methods of screening for a compound that improves the ability of biological material to survive anoxia or hypoxia or to undergo stasis comprising: a) incubating a first biological material capable of undergoing stasis under hypoxic or anoxic conditions; b) exposing the first biological material to a candidate compound; c) incubating a second biological material capable of undergoing stasis under the same hypoxic conditions as the first biological material; d) comparing the first biological material and the second biological material. A candidate compound is one that improves the ability of a biological material to survive under anoxic or hypoxic conditions. Any of the embodiments described herein may be applied to practice any of the screening methods of the invention. The invention includes the use of biological material that is capable of undergoing stasis during a particular point in its development or lifetime, but is not capable of undergoing stasis at the time of testing. Stasis inducer compounds, including reversible stasis inducer compounds (compounds that induce reversible stasis) identified by screening methods form part of the present invention. Methods of the invention include using the identified compounds. Thus, the present invention includes methods of inducing stasis in biological material by administering to the biological material an effective amount of a stasis inducer compound identified by processes described herein. It also includes methods of treating a tumor or inhibiting its growth using the antitumor compounds of the claimed invention. Such compounds may be formulated in pharmaceutically acceptable formulations and administered to a tumor cell or to a patient using routine routes of administration. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. detailed-description description="Detailed Description" end="lead"? |
Braca1/acc alpha molecular complexes, diagnostic and therapeutic applications |
The invention concerns a molecular complex comprising: a first polypeptide including the sequence of amino acids 1640 to 1863 the human BRCA1 protein or a similar sequence of amino acids of the protein BRCA1 in another animal species, and a second polypeptide including a fragment of the ACC-α protein capable of binding with said first protein. |
1-10. (canceled) 11. A molecular complex comprising: a first polypeptide comprising a sequence of amino acids 1640 to 1863 of human BRCA1 protein or the same sequence of amino acids of the BRCA1 protein in another species of animal, and a second polypeptide comprising a fragment of ACC-α protein capable of binding the said first polypeptide. 12. A method of screening in vitro of molecules useful for prevention or treatment of cancer of breast and/or of ovary in which molecules are tested for their capacity to modulate interaction between BRCA1 and ACC-α proteins. 13. The method of screening according to claim 12, comprising: a) contacting, in any order whatsoever, two different partners or three different partners selected from the group consisting of a first polypeptide comprising the sequence of amino acids 1640 to 1863 of the human BRCA1 protein or the same sequence of amino acids of the BRCA1 protein, a second polypeptide comprising a fragment of the ACC-α protein capable of binding the said first polypeptide, and a candidate molecule; b) incubating said partners for a sufficient time to permit any interaction thereof; c) in the case where only two different partners have been selected at step a), adding the third partner selected from the said group and incubating for a sufficient time to permit any interaction thereof; d) determining the capacity of the candidate molecule to modulate the interaction between BRCA1 and ACC-α, wherein said capacity is indicative of a molecule useful for the prevention or the treatment of cancer of the breast and/or of the ovary. 14. The method of screening according to claim 12, comprising: a) contacting a first polypeptide comprising the sequence of amino acids 1640 to 1863 of the human BRCA1 protein or the same sequence of amino acids of the BRCA1 protein with a second polypeptide comprising a fragment of the ACC-α protein capable of binding the said first polypeptide, one at least of the polypeptides being labelled in a detectable manner; b) adding a candidate molecule capable of modulating the interaction; c) incubating the said polypeptides in the presence of the candidate molecule under conditions and for a period of time which are sufficient for the binding between the said polypeptides to take place; d) quantifying the number of labelled molecules bound in the presence of increasing concentrations of the candidate molecule, wherein a modification of the number of labelled molecules bound with increasing concentrations of the candidate molecule is indicative of a molecule useful for the prevention or the treatment of cancer of the breast and/or of the ovary. 15. The method of screening according to claim 12, wherein said molecule capable of modulating the BRCA1/ACC-α interaction is an agonist. 16. The method of screening according to claim 12, wherein the said molecule capable of modulating the BRCA1/ACC-α interaction is an antagonist. 17. A method of identification ex vivo of molecules constituting endogenous ligands of a BRCA1/ACC-α complex comprising: a) contacting a BRCA1/ACC-α complex with a biological specimen under conditions permitting the formation of interactions between the complex and any ligands; b) detecting interactions between the BRCA1/ACC-α complex and the said ligands; and c) identifying ligands interacting with the complex as molecules constituting endogenous ligands of the BRCA1/ACC-α complex. 18. A method of in vitro diagnosis of a predisposition to cancer of the breast and/or of the ovary, comprising determining a modification of a BRCA1/ACC-α interaction in a subject relative to a control population, the modification of the BRCA1/ACC-α interaction being associated with a variation of the risk of developing a cancer of the breast and/or of the ovary. 19. An anti-human ACC-α antibody directed against peptides of sequence SEQ ID No. 1 and SEQ ID No. 2. 20. An antibody directed against a BRCA1/ACC-α molecular complex, which does not interact with the BRCA1 or ACC-α proteins alone. |
Method and measuring changes in microvascular capillary blood flow |
A method of measuring changes in microvascular capillary flow in a predetermined region in a subject comprising: (a) administering an ultrasound contrast medium to said subject such that said contrast medium reaches the microvascular capillaries in said predetermined region; (b) measuring microvascular capillary blood flow volume and/or microvascular flow index of said capillaries; (c) applying a defined signal to said subject; and, (d) measuring changes in said microvascular capillary flow. |
1. A method of measuring changes in microvascular capillary flow in a predetermined region in a subject comprising: (a) administering an ultrasound contrast medium to said subject such that said contrast medium reaches the microvascular capillaries in said predetermined region; (b) measuring microvascular capillary blood flow volume and/or microvascular flow index of said capillaries; (c) applying a defined signal to said subject; and, (d) measuring changes in said microvascular capillary flow. 2. A method according to claim 1 wherein the measurement is made by ultrasound imaging. 3. A method according to claim 1 wherein said contrast medium is microbubbles. 4. A method according to claim 3 wherein said microbubbles are selected from albumin and/or phospholipid microbubbles. 5. A method according to claim 2 wherein said ultrasound image may be obtained by using contrast enhanced ultrasound. 6. A method according to claim 1 wherein said defined signal includes any signal potentially or actually capable of affecting microvascular capillary flow. 7. A method according to claim 6 wherein said defined signal includes any one or a combination of: applying insulin to the capillary region of a subject; applying a drug intended to increase or decrease microvascular capillary flow of a subject; and causing a defined level of exercise to be applied to a muscle area associated with the capillaries of said subject. 8. A diagnostic method for differentiating normal from abnormal responses in microvascular capillaries to the application of a defined signal, said method comprising the use of the method of claim 1 in the following steps: (a) measuring microvascular capillary flow in a predetermined region in a subject in response to said defined signal; (b) measuring microvascular capillary flow in an analogous predetermined region in a control subject; (c) comparing said flow rates. 9. A method of diagnosis characterised by the direct measurement of any change in microvascular capillary flow at the source in a subject using the methods of claim 1. 10. A method for determining changes in nutritive capillary recruitment in a subject comprising the use of the method of claim 1 in the following steps: (a) measuring microvascular capillary flow in a subject in a first situation; (b) measuring microvascular capillary flow in a subject in a second situation; (c) comparing said flow rate. 11. A method of diagnosis of any one or a combination of conditions including Type I or Type II diabetes, hypertension, obesity and patients in critical care comprising the following steps: (a) administering an ultrasound contrast medium to a subject in need of diagnosis such that said contrast medium reaches the microcapillaries of said subject; (b) measuring microvascular capillary blood flow volume and br microvascular flow velocity index of said capillaries; (c) applying a defined signal to said subject; (d) measuring changes to said microvascular capillary flow wherein said measurement is made by ultrasound imaging; (e) repeating steps (a) to (d) in a control subject and/or comparing know control result, such that any variation of results from the control indicates the existance of predisposition of said subject to said condition. 12. A method according to claim 11 wherein said defining signal is insulin. 13. A method according to claim 1 substantially as herein before described with reference to the examples. |
<SOH> BACKGROUND OF THE INVENTION <EOH>A number of laboratories have now reported an effect of insulin to increase total blood flow to muscle and that this effect is impaired in states of insulin resistance (1-3). However, the role of the increase in total blood flow mediated by insulin is controversial. Several research groups claim that insulin-mediated changes in total blood flow relate poorly to muscle glucose uptake under a number of circumstances, including insulin dose and time course (4-6). In addition there have been studies where total flow changes persist when glucose uptake is inhibited (7,8). Also, most vasodilators that augment total blood flow to the limbs do not enhance insulin action nor do they overcome insulin resistance (9, 10). The applicants have shown that hormone and nutrient access by regulating flow between nutritive and non-nutritive flow routes in muscle is a central process in controlling both muscle metabolism and function. It has also been shown that restriction of insulin and glucose access by pharmacologically manipulating flow to be predominantly non-nutritive, creating a state of insulin resistance. The applicants then searched for a method that could detect changes in the proportion of nutritive (capillary) to non-nutritive blood flow in this tissue that might have application in vivo and ultimately to humans. Marker enzymes located in one or other of the two vascular networks (nutritive or non-nutritive) were to provide the key. Thus a first method was developed which involved 1-methylxanthine (1-MX) (20), as an exogenous substrate for xanthine oxidase, an enzyme shown by others to reside predominantly in capillary (nutritive) endothelial cells (21). 1-MX was infused intra-arterially and its metabolite 1-methyl urate measured in venous blood by HPLC; the extent of conversion a reflection of capillary exposure. Characterization under a number of conditions revealed that 1-MX metabolism was indeed directly proportional to nutritive, or capillary flow, which in the constant-flow perfused hindlimb system could be altered by applying various vasoconstrictors or by simulating exercise. The 1-MX method was tested in vivo using the hyperinsulinaemic euglycaemic clamp in rats and it was shown for the first time that insulin acted directly to recruit capillary flow in muscle. Further work showed that pharmacological manipulation to decrease the proportion of nutritive blood flow by an infused vasoconstrictor, created a state of insulin resistance. These latter findings directly linked blood pressure through blood redistribution to insulin resistance in vivo—a situation that is evident from a number of epidemiological studies of human populations. In addition, a close link between capillary recruitment and muscle glucose uptake began to emerge from these and previous 1-MX studies. A second method was devised using the latest technologies in ultrasound. The ultrasound method relies on the increased echogenicity of microbubbles which are continuously infused intravenously during data acquisition. The acoustic signal that is generated from the microbubbles when exposed to ultrasound produces tissue opacification which is proportional to the number of microbubbles within the ultrasound beam. Using harmonic pulsing methods essentially all microbubbles within the ultrasound beam are destroyed in response to a single pulse of high-energy ultrasound and an image is obtained. In the time interval between subsequent pulsing episodes, microbubbles flowing into the tissue are replenished within the beam and affect the intensity of the signal from the next high energy pulse. Repeating this process with pulse delays between 50 msec and 20 sec, the beam will be fully replenished and further increases in the time between each pulsing interval will not produce a change to tissue opacification. The rate of microbubble reappearance within the ultrasound beam provides an indication of capillary velocity and the degree of tissue opacification provides a measurement of capillary blood volume (CBV). Images are background-subtracted from images from a pulsing interval of 1000 ms which represents the replenishment of arteries and arterioles thus providing a measurement of capillary flow. The plateau tissue opacification (measured as videointensity) is the determination of capillary blood volume. Using this approach, changes in capillary blood volume in response to insulin and exercise were assessed in the skeletal muscle of the rat hindlimb in vivo and compared to data obtained using 1-MX metabolism. Compared to baseline values, saline-infusion resulted in little change in capillary blood volume whereas marked increases in capillary blood volume occurred during euglycemic insulin clamp (3 mU/min/kg). Preliminary studies demonstrated that Contrast Enhanced Ultrasound (CEU) data correlates well with 1-MX metabolism data, and that capillary blood volume increases 2-3 fold during these physiologic doses of insulin. A particular advantage of the ultrasound method is that it is relatively non-invasive and is suitable for human use thereby opening up possibilities for its use in diagnosis and the monitoring of outcomes from therapeutic interventions. Therefore, from these studies a routine diagnostic test emerges where impaired response to a signal, for example insulin, in terms of capillary recruitment can be made. This would take the form of measuring the response to a number of possible stimuli that would be expected to lead to an increased signal reflecting capillary recruitment. For insulin the stimuli may include: the response to a standard meal; insulin infusion; insulin injection; insulin inhalation; oral insulin releasing drugs; oral insulin enhancing drugs; or a combination of the aforementioned; or exercise. Patients where the techniques and methods of the invention outlined above would be applicable include those with type 2 diabetes, type 1 diabetes, hypertension, obesity, a combination of the aforementioned and critical care patients. |
<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect the invention provides a method of measuring changes in microvascular capillary flow in a predetermined region in a subject comprising: a) administering an ultrasound contrast medium to said subject such that said contrast medium reaches the microvascular capillaries in said predetermined region;. b) measuring microvascular capillary blood flow volume and/or microvascular flow velocity index of said capillaries; c) applying a defined signal to said subject; d) measuring changes to said microvascular capillary flow wherein the measurement may be made by ultrasound imaging or other methods. The ultrasound contrast medium may be microbubbles and is most preferably albumin microbubbles, although other contrast media may be used including microbubbles of similar size (3-4 micrometres diameter) and echo characteristics. e.g. phospholipid microbubbles. The ultrasound image may be obtained by using contrast enhanced ultrasound. The defined signal can be any thing potentially or actually affecting microvascular capillary flow; including insulin applied to said capillaries, a drug intended to increase or decrease microvascular capillary flow or a defined level of exercise applied to a muscle area associated with said capillaries. In another aspect the invention provides a diagnostic method to differentiate normal from abnormal responses in microvascular capillaries to the application of a defined signal comprising the use of the above method to measure microvascular capillary flow in a predetermined region in a subject in response to said defined signal when compared to the flow in a control microvascular capillary region. In another aspect the invention provides a diagnostic method incorporating the direct measurement of change in microvascular capillary flow at the source using the measurement methods as previously set out. In another aspect the invention provides a method for determining changes in nutritive capillary recruitment using the measurement methods as previously set out. In another aspect the invention provides a method of diagnosis of any one or a combination of conditions including Type 1 &2 diabetes, hypertension, obesity and critical care patients comprising: a) administering an ultrasound contrast medium to a subject in need of diagnosis such that said contrast medium reaches said microcapillaries; b) measuring microvascular capillary blood flow volume and/or microvascular flow velocity index of said capillaries; c) applying a defined signal to said subject; d) measuring changes to said microvascular capillary flow wherein the measurement is made by ultrasound imaging; e) repeating steps (a) to (d) in a control subject and/or comparing known control results, such that any variation of results from the control indicates the existence or predisposition of said subject to said condition. The defined signal may be insulin. detailed-description description="Detailed Description" end="lead"? |
Determination of bone-sialoprotein in bodily fluids for oncological problems |
The invention relates to antibodies or a plurality of antibodies against human bone sialoprotein (BSP), characterized in that the antibodies bind to epitopes which are present only in human bone sialoprotein from tumor cells, the post-translational glycosylation of which is modified or incomplete in the region of amino acids of 120 to 135, containing the amino acids TGLAA (SEQ ID NO: 2), in comparison with normal bone sialoprotein from bones. The antibodies are put to use in an immunoassay for the diagnosis and prognosis of tumor diseases, in particular the diagnosis and prognosis of bone metatstases in the case of primary breast carcinoma. |
1. One or more antibodies against human bone sialoprotein (BSP), that specifically bind epitopes which are present in human bone sialoprotein from tumor cells, the post-translational glycosylation of which is modified or incomplete in the region of amino acids of 120 to 135 (SWISSPROT:SIAL_HUMAN, Acc. No. P21815, without signal sequence), containing the amino acids TGLAA (SEQ ID NO: 2), in comparison with normal bone sialoprotein from bones. 2. Antibodies according to claim 1, produced with a bone sialoprotein modified in its glycosylation, chemically or naturally, as antigen. 3. Antibodies according to claim 1 or 2, produced with a bone sialoprotein from tumor cells as antigen. 4. Antibodies according to claim 3, whereby the bone sialoprotein modified in its glycosylation is produced by genetic engineering in tumor cells. 5. Antibodies according to claim 1, produced against a peptidic antigen, including the amino acid sequence TGLAA (SEQ ID NO: 2) or YTGLAA (SEQ ID NO: 3), if appropriate coupled to the carrier protein. 6. Antibodies according to claim 2, wherein there is employed bone sialoprotein, modified in its glycosylation, from bone material the donor of which was not capable of normal glycosylation of bone proteins. 7. Antibodies according to claim 1, 5 or 6, which are IgY antibodies of the chicken. 8. Antibodies according to claim 1, 5 or 6, which are human or humanized. 9. A method for the determination of bone sialoprotein from tumor cells in body fluids, comprising contacting a sample from said body fluid with antibodies according to claim 1, 5 or 6 and detecting binding of said antibodies to bone sialoprotein from said sample. 10. The method according to claim 9 for the diagnosis and prognosis of bone metastases. 11. (canceled). 12. A pharmaceutical composition comprising as active ingredient antibodies according to claim 1, 5 or 6. 13-14. (canceled). |
Low-voltage circuit-breaker with an electronic overload trip and trip magnets |
A low voltage circuit breaker includes an electronic trip and trip magnets. A trip magnet is controlled by an overload trip and a further trip magnet, such as a low voltage trip or a working current trip. The trip magnets are mounted on a rear face, facing away from the operating front of the electronic trip. In the same section of the operating field, display and reset devices are accessible to the user. |
1. A low-voltage power breaker, comprising: an electronic overcurrent release; and tripping magnets, adapted to trip a latch when at least one of the electronic overcurrent release and a special signal causes closed switching contacts to be released, the electronic overcurrent release and the tripping magnets being fit together to form a protective module, wherein the electronic overcurrent release includes a front control panel, accessible via a control panel of the power breaker, and wherein the tripping magnets are fit to the electronic overcurrent release on a side of a housing of the electronic overcurrent release that is remote from the front control panel. 2. The low-voltage power breaker as claimed in claim 1, wherein the tripping magnets include at least one of display and reset elements, visible on the front control panel of the electronic overcurrent release. 3. A module of a circuit breaker, comprising: an electronic overcurrent release; and tripping magnets, adapted to trip a latch, when at least one of the electronic overcurrent release and a special signal causes closed switching contacts to be released, wherein the electronic overcurrent release includes a front control panel, accessible via a control panel of the power breaker, and wherein the tripping magnets are located on a side of a housing of the electronic overcurrent release that is remote from the front control panel. 4. The module as claimed in claim 3, wherein the tripping magnets include at least one of display and reset elements, visible on the front control panel of the electronic overcurrent release. 5. A low-voltage power breaker, comprising: an electronic overcurrent release; and means for tripping a latch when at least one of the electronic overcurrent release and a special signal causes closed switching contacts to be released, the electronic overcurrent release and the means for tripping being fit together to form a protective module, wherein the electronic overcurrent release includes a front control panel, accessible via a control panel of the power breaker, and wherein the means for tripping is located on a side of a housing of the electronic overcurrent release that is remote from the front control panel. 6. The low-voltage power breaker as claimed in claim 5, wherein the means for tripping include at least one of display and reset elements, visible on the front control panel of the electronic overcurrent release. |
<SOH> BACKGROUND OF THE INVENTION <EOH>A power breaker has been disclosed in U.S. Pat. No. 4,700,161. This power breaker has a known compact design having a molded case and a toggle lever drive (MCCB). In order to make it possible to replace or refit modules of the tripping magnets, access can be gained to said tripping magnets via the front control panel of the power breaker once a cover has been removed. The electronic overcurrent release does not have its own control panel owing to the compact design of the power breaker. Another feature of the design of compact power breakers is the fact that the operator can detect a tripping procedure not by means of a special display element but by means of a specific position of the outer handle of the toggle lever drive. The resetting required for reconnection of the power breaker also takes place by means of the handle of the toggle lever drive. Electronic overcurrent releases and tripping magnets are correspondingly used in the same manner in low-voltage power breakers of open design (PCBs) which are distinguished from the design with the toggle lever drive by their suitability for higher rated currents and by their higher switching capacity as well as by a storage drive. The overcurrent releases of these power breakers also make it possible to set all the important parameters in order to be able to match the protective action of the power breaker, for example, to the properties of a load. For this purpose, the overcurrent releases are provided with their own front control panel which is included in the control panel of the power breaker and at which access can be gained to the setting elements. Resetting, following tripping, takes place in this case by means of a special element which is arranged in the vicinity of the front control panel of the overcurrent release (Catalog 3WN6, 1995, page 2, Siemens Publication E20002-K1801-B401-A1). |
<SOH> SUMMARY OF THE INVENTION <EOH>An embodiment of the invention may be based on an object, in a power breaker, of accommodating tripping magnets in a space-saving manner. Preferably, their display and reset elements are arranged on the front control panel of the power breaker such that they can be seen. An object may be achieved according to an embodiment of the invention by the electronic overcurrent release having, in a manner known per se, a front control panel which is accessible via a control panel of the power breaker, and/or by the tripping magnets being fit to the overcurrent release on the side of a housing of the latter that is remote from the front control panel. Using an arrangement which is the opposite of that disclosed in U.S. Pat. No. 4,700,161, mentioned initially,—tripping magnets underneath or behind the electronic overcurrent release—the user may operate the electronic overcurrent release in the same manner whilst retaining the advantage of the combined design of the two assemblies. On the other hand, the display and operating elements of the tripping magnets are now part of the front control panel of the overcurrent release, which improves clarity and thus operational reliability. An important factor here is the fact that the combination of all of the tripping magnets provided to form a assembly fit to the overcurrent release accordingly also makes two, or even more, display and reset elements visible and accessible to the operator on the front control panel of the overcurrent release. |
Statistical model |
A method of building a statistical shape model by automatically establishing correspondence between a set of two dimensional shapes or three dimensional shapes, the method comprising: a) determining a parameterisation of each shape, building a statistical shape model using the parameterisation, using an objective function to provide an output which indicates the quality of the statistical shape model; performing step a) repeatedly for different parameterisations and comparing the quality of the resulting statistical shape models using output of the objective function to determine which parameterisation provides the statistical shape model having the best quality, wherein the output of the objective function is a measure of the quantity of information required to code the set of shapes using the statistical shape model. |
1. A method of building a statistical shape model by automatically establishing correspondence between a set of two dimensional shapes or three dimensional shapes, the method comprising: a. determining a parameterisation of each shape, building a statistical shape model using the parameterisation, using an objective function to provide an output which indicates the quality of the statistical shape model, b. performing step a repeatedly for different parameterisations and comparing the quality of the resulting statistical shape models using output of the objective function to determine which parameterisation provides the statistical shape model having the best quality, wherein the output of the objective function is a measure of the quantity of information required to code the set of shapes using the statistical shape model. 2. A method according to claim 1, wherein the parameterisation used to perform step a are selected using an optimization method based upon previous parameterisations and the quality of the statistical shape models generated using those parameterisations as indicated by the output of the objective function. 3. A method according to claim 2, wherein the output of the objective function comprises a value indicative of the amount of information required to code parameters of the model, and a value indicative of the information required to code residuals for the set of shapes. 4. A method according to claim 3, wherein the output of the objective function further comprises a value indicative of the amount of information required to code mapping between parameters and the shapes. 5. A method according to claim 4 wherein for a given parameterisation the objective function provides a single scalar output. 6. A method according to claim 5 wherein the single scalar output comprises a combination of the value indicative of the amount of information required to code parameters of the model, the value indicative of the information required to code residuals for the set of shapes, and the value indicative of the amount of information required to code mapping between parameters and the shapes. 7. A method according to claim 4, wherein the mapping comprises a mean vector and a covariance matrix. 8. A method according to claim 3 wherein the parameters of the model are parameter vectors, and the average amount of information per parameter vector is determined and multiplied by the number of shapes in the training set, to provide the value indicative of the amount of information required to code parameters of the model. 9. A method according to claim 3 wherein the residuals are residual vectors, and the average amount of information per residual vector is determined and multiplied by the number of shapes in the training set, to provide the value indicative of the information required to code residuals for the complete set of training shapes. 10. A method according to claim 9, wherein the number of residuals in each residual vector is twice the number of parameterisations used to generate the model. 11. A method according to claim 1, wherein the number of modes of variation of the model is selected to represent the training set to a given accuracy. 12. A method according to claim 1, wherein the boundary of each two-dimensional shape of the training set is recursively subdivided by inserting landmarks the parameterisation of the boundary being represented by the position of each landmark as a fraction of boundary path length between preceding and following landmarks. 13. A method according to claim 12, wherein the parameterisation which provides the best optimisation value is determined using a stochastic optimisation method. 14. A method according to claim 1 wherein a set of curves is used to determine a parameterisation function for the training set. 15. A method according to claim 14, wherein the set of curves is a cumulative density function. 16. A method according to claim 15, wherein the cumulative density function comprises a sum of kernel curves. 17. A method according to claim 16, wherein the cumulative density function is determined by combining increasing numbers of kernel curves to provide increasing resolution levels, and the parameterisation function which provides the statistical shape model of the best quality is determined using the output of the objective function for each resolution level. 18. A method according to claim 16, wherein the kernel curves are Gaussian functions. 19. A method according to claim 16 wherein the kernel curves are Cauchy functions. 20. A method according to claim 16 wherein the kernel curves are two dimensional curves. 21. A method according to claim 16 wherein the kernel curves are three dimensional curves. 22. A method according to claim 1 wherein the boundary of each shape of the training set is parameterised by specifying a set of control landmarks used to determine a parameterisation function. 23. A method according to claim 22, wherein the parameterisation function is determined for increasing numbers of control landmarks to provide increasing resolution levels, and the parameterisation function which provides the statistical shape model of the best quality is determined using the output of the objective function for each resolution level. 24. A method according to claim 22 wherein equally spaced landmarks are provided between the control landmarks. 25. A method according to claim 1, wherein the shape and grey-level appearance are both included in model. 26. A method of parameterising a set of three dimensional shapes, the method comprising: a. mapping each three dimensional shape to a simple canonical form of an appropriate topology, b. applying a set of landmarks to the simple form for a given level of resolution, each landmark being constrained to lie inside a spherical triangle formed by a triple of landmark points of a lower resolution level, c. upon the completion of the application of the set of landmarks for the resolution level, projecting the set of landmarks onto the three dimensional shapes, d. building a statistical shape model using the set of landmarks of the resolution level, e. using an objective function to provide an output which indicates the quality of the statistical shape model determined using the set of landmarks of the resolution level, f. comparing the output with output determined using a different set of landmarks having the same level of resolution, and g. repeating steps a to f to determine which set of landmarks which provides the best quality of statistical shape model, h. repeating steps a to g at a higher level of resolution. 27. A method of parameterising a set of three dimensional shapes, the method comprising: a. mapping each three dimensional shape to simple canonical form of an appropriate topology, b. applying a set of landmarks to the simple form, c. modifying the locations of the landmarks by applying a transformation, d. projecting the set of landmarks onto the three dimensional shapes, e. building a statistical shape model using the set of landmarks, f. using an objective function to provide an output which indicates the quality of the statistical shape model determined using the set of landmarks, g. comparing the output with output determined using landmarks having different locations, and h. repeating steps c to g to determine which set of landmarks which provides the best quality of statistical shape model. 28. A method according to claim 27, wherein the transformation comprises for a selected landmark pushing other landmarks away from the selected landmark along the surface of the simple form, or pulling other landmarks towards the selected landmark along the surface of the simple form. 29. A method according to claim 28, wherein the transformation is defined by a wrapped kernel. 30. A method according to claim 29, wherein the wrapped kernel is a warpped Cauchy distribution. 31. A method according to claim 29, wherein the wrapped kernel is a wrapped Gaussian distribution. 32. A method according to claim 28 wherein the simple form is a sphere or a torus. |
Novel surface explosed immunoglobulin d-binding protein from foraxella catarrhalis |
The present invention relates to a surface exposed protein, which can be detected in Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, to an immunogenic or IgD-binding fragment of said surface exposed protein, and to an immunogenic and adhesive fragment of said surface exposed protein. DNA segments, vaccines, plasmids and phages, non human hosts, recombinant DNA molecules and plants, fusion proteins and polypeptides and fusion products are also described. A method of detecting IgD, a method of separating IgD, a method of isolation of a surface exposed protein of Moraxella catarrhalis and a method for treatment of an autoimmune disease are also disclosed. |
1. A surface exposed protein, which can be detected in Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, or an immunogenic and adhesive fragment of said surface exposed protein. 2. An immunogenic or IgD-binding fragment of a surface exposed protein as defined in claim 1, which fragment can be detected in Moraxella catarrhalis, having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof. 3. An immunogenic or IgD-binding fragment according to claim 2, having an amino acid sequence as described in SEQ ID NO 10. 4. An immunogenic and adhesive fragment of a surface exposed protein as defined in claim 1, which fragment can be detected in Moraxella catarrhalis, having a capacity of binding erythrocytes and epithelial cells. 5. An immunogenic and adhesive fragment according to claim 4, having an amino acid sequence as described in SEQ ID NO 8. 6. A DNA segment comprising a DNA sequence, as shown in SEQ ID NO 2, which DNA sequence codes for a surface exposed protein of Moraxella catarrhalis as defined in claim 1, or naturally occurring or artificially modified variants of said DNA sequence. 7. A DNA segment comprising a DNA sequence which codes for an immunogenic or IgD-binding fragment as defined in claim 2. 8. A DNA segment according to claim 7, comprising a DNA sequence, as shown in SEQ ID NO 11, which DNA sequence codes for an immunogenic or IgD-binding fragment having an amino acid sequence as described in SEQ ID NO 10. 9. A DNA segment comprising a DNA sequence, which codes for an immunogenic and adhesive fragment of a surface exposed protein as defined in claim 4. 10. A DNA segment according to claim 9, comprising a DNA sequence, as shown in SEQ ID NO 9, which DNA sequence codes for an immunogenic and adhesive fragment which can be detected in Moraxella catarrhalis having a capacity of binding erythrocytes and epithelial cells and an amino acid sequence as described in SEQ ID NO 8. 11. A vaccine containing a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, or an immunogenic and adhesive fragment of said surface exposed protein. 12. A vaccine containing an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis, having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment. 13. A vaccine according to claim 12 containing an immunogenic or IgD-binding fragment which can be detected in Moraxella catarrhalis having a capacity of selectively binding membrane bound or soluble IgD or naturally occurring or artificially modified variants thereof and an amino acid sequence as described in SEQ ID NO 10. 14. A vaccine containing an immunogenic and adhesive fragment of a surface exposed protein of Moraxella catarrhalis as defined in claim 4. 15. A vaccine according to claim 14 containing an immunogenic and adhesive fragment of a surface exposed protein which can be detected in Moraxella catarrhalis having a capacity of binding erythrocytes and epithelial cells and an amino acid sequence as described in SEQ ID NO 8. 16. A vaccine according to claim 11, combined with another vaccine. 17. A vaccine according to claim 11, combined with an immunogenic portion of another molecule. 18. A plasmid or phage comprising a DNA sequence, which codes for a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants. 19. A plasmid or phage comprising a DNA sequence, which codes for an immunogenic or IgD-binding fragment of a surface exposed protein as defined in claim 1, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment. 20. A plasmid or phage according to claim 19 comprising a DNA sequence, which codes for an immunogenic or IgD-binding fragment as which can be detected in Moraxella catarrhalis having a capacity of selectively binding membrane bound or soluble IgD or naturally occurring or artificially modified variants thereof and an amino acid sequence as described in SEQ ID NO 10. 21. A plasmid or phage comprising a DNA sequence, which codes for an immunogenic and adhesive fragment of a surface exposed protein as defined in claim 1, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells or naturally occurring or artificially modified variants of said fragment. 22. A plasmid or phage according to claim 21 comprising a DNA sequence, which codes for an immunogenic and adhesive fragment of a surface exposed protein which can be detected in Moraxella catarrhalis having a capacity of binding erythrocytes and epithelial cells and an amino acid sequence as described in SEQ ID NO 8. 23. A non human host comprising at least one plasmid or phage as defined in claim 18, and capable of producing said protein or variants, or said immunogenic or IgD-binding fragment of said protein or variants, or said immunogenic and adhesive fragment of said protein, which host is chosen among bacteria, yeast and plants. 24. A host according to claim 23, which is E. coli. 25. A recombinant DNA molecule comprising a DNA sequence coding for a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, which DNA sequence is fused to another gene. 26. A recombinant DNA molecule comprising a DNA sequence coding for an immunogenic or IgD-binding fragment of a surface exposed protein as defined in claim 1, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, which DNA sequence is fused to another gene. 27. A recombinant DNA molecule according to claim 26 comprising a DNA sequence, which codes for an immunogenic or IgD-binding fragment which can be detected in Moraxella catarrhalis having a capacity of selectively binding membrane bound or soluble IgD or naturally occurring or artificially modified variants thereof and an amino acid sequence as described in SEQ ID NO 10 which DNA sequence is fused to another gene. 28. A recombinant DNA molecule comprising a DNA sequence coding for an immunogenic and adhesive fragment of a surface exposed protein as defined in claim 1, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, which DNA sequence is fused to another gene. 29. A recombinant DNA molecule according to claim 28 comprising a DNA sequence, which codes for an immunogenic and adhesive fragment of a surface exposed protein which can be detected in Moraxella catarrhalis having a capacity of binding erythrocytes and epithelial cells and an amino acid sequence as described in SEQ ID NO 8 which DNA sequence is fused to another gene. 30. A plasmid or phage comprising a fused DNA sequence as defined in claim 25. 31. A non-human host comprising at least one plasmid or phage as defined in claim 30, which host is chosen among bacteria, yeast and plants. 32. A host according to claim 31, which is E. coli. 33. A fusion protein or polypeptide, in which a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, is combined with another protein by the use of a recombinant DNA molecule as defined in claim 25. 34. A fusion protein or polypeptide in which an immunogenic or IgD-binding fragment of a surface exposed protein as defined in claim 1, which fragment can be detected in Moraxella catarrhalis, having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, is combined with another protein by the use of a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic or IgD-binding fragment of a surface exposed protein which can be detected in Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, or an immunogenic and adhesive fragment of said surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, which DNA sequence is fused to another gene. 35. A fusion protein or polypeptide according to claim 34 in which an immunogenic or IgD-binding fragment which can be detected in Moraxella catarrhalis having a capacity of selectively binding membrane bound or soluble IgD or naturally occurring or artificially modified variants thereof and an amino acid sequence as described in SEQ ID NO 10 is combined with another protein by the use of a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic or IgD-binding fragment of a surface exposed protein which can be detected in Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, or an immunogenic and adhesive fragment of said surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, which DNA sequence is fused to another gene. 36. A fusion protein or polypeptide in which an immunogenic and adhesive fragment of a surface exposed protein as defined in claim 1, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, is combined with another protein by the use of a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic and adhesive fragment of said surface exposed protein which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, which DNA Sequence is fused to another gene. 37. A fusion protein or polypeptide according to claim 36 in which an immunogenic and adhesive fragment which can be detected in Moraxella catarrhalis having a capacity of binding erythrocytes and epithelial cells and an amino acid sequence as described in SEQ ID NO 8 is combined with another protein by the use of a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic and adhesive fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment and an amino acid sequence as described in SEQ ID NO 8, which DNA sequence is fused to another gene. 38. A fusion product, in which a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, is covalently, or by any other means, bound to a protein, carbohydrate or matrix. 39. A fusion product in which an immunogenic or IgD-binding fragment of a surface exposed protein as defined in claim 2, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, is covalently, or by any other means, bound to a protein, carbohydrate or matrix. 40. A fusion product according to claim 39 in which an immunogenic or IgD-binding fragment having an amino acid sequence as described in SEQ ID NO 10 is covalently, or by any other means, bound to a protein, carbohydrate or matrix. 41. A fusion product in which an immunogenic and adhesive fragment of a surface exposed protein as defined in claim 2, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, is covalently, or by any other means, bound to a protein, carbohydrate or matrix. 42. A fusion product according to claim 41 in which an immunogenic and adhesive fragment which can be detected in Moraxella catarrhalis having a capacity of binding erythrocytes and epithelial cells and an amino acid sequence as described in SEQ ID NO 8 is covalently, or by any other means, bound to a protein, carbohydrate or matrix. 43. A method of detecting IgD using a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, optionally labelled and/or bound to a matrix. 44. A method of detecting IgD using an immunogenic or IgD-binding fragment of a surface exposed protein as defined in claim 2, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally labelled and/or bound to a matrix. 45. A method of detecting IgD according to claim 44 using an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof and having an amino acid sequence as described in SEQ ID NO 10, and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, optionally labelled and/or bound to a matrix. 46. A method of separating IgD using a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, optionally bound to a matrix. 47. A method of separating IgD using an immunogenic or IgD-binding fragment of a surface exposed protein as defined in claim 2, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally bound to a matrix. 48. A method of separating IgD according to claim 47 using an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof and having an amino acid sequence as described in SEQ ID NO 10, having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, optionally labelled and/or bound to a matrix. 49. A method of isolation of a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, said method comprising the steps: a) subjecting a suspension of Moraxella catarrhalis to an extraction process by adding a zwitterionic or non-ionic detergent, optionally in the presence of EDTA; b) applying the extract comprising the IgD-binding protein of Moraxella catarrhalis from step a) to an ad-sorption column; c) eluating the IgD-binding protein; and d) separating the IgD-binding protein. 50. A method according to claim 49, wherein the detergent is selected from the group comprising Empigen®, n-Octyl-p-D glucoside and Triton X-100+0.01M EDTA. 51. A method according to claim 49, wherein the concentration of the detergent in step a) is within the range of 0.1-5%. 52. A method for treatment of an autoimmune disease comprising extra corporal circulation of the blood through a material comprising a surface exposed protein as defined in claim 1, for removal of IgD from the blood. 53. A purified antibody which is specific to an immunogenic portion of a surface exposed protein Moraxella catarrhalis, said protein having an amino acid sequence as described in SEQ ID NO 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants. 54. A purified antibody according to claim 48, which is specific to an immunogenic or IgD-binding fragment having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof and having an amino acid sequence as described in SEQ ID NO 10, having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment. 55. A purified antibody according to claim 48, which is specific to an immunogenic or adhesive fragment having an amino acid sequence as described in SEQ ID NO 8, having a capacity of binding erythrocytes and epithelial cells. 56. A method according to claim 50, wherein the concentration of the detergent in step a) is within the range of 0,1-5%. 57. A method according to claim 49 wherein the concentration of the detergent of step a) is about 3%. 58. A method according to claim 50 wherein the concentration of the detergent of step a) is about 3%. 59. A method for treatment of an autoimmune disease comprising extra corporal circulation of the blood through a material comprising an immunogenic or IgD fragment of a surface exposed protein as defined in claim 2 for removal of IgD from the blood. 60. A method for treatment of an autoimmune disease comprising extra corporal circulation of the blood through a material comprising an immunogenic or IgD fragment of a surface exposed protein as defined in claim 3 for removal of IgD from the blood. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Moraxella catarrhalis is a Gram-negative diplococcus that for a long time was considered a relatively harmless commensal in the respiratory tract. At present, it is the third most frequent cause of otitis media and also a significant agent in sinusitis and lower respiratory tract infections in adults with pulmonary disease. M. catarrhalis is also one of the most common inhabitants of the pharynx of healthy children. Two decades ago, Haemophilus influenzae and M. catarrhalis were shown to display a strong affinity for both soluble and surface-bound human IgD (1). The IgD-binding seems to be paralleled by a similar interaction with surface-bound IgD at the cellular level, a phenomenon that explains the strong mitogenic effects on human lymphocytes by H. influenzae and M. catarrhalis (2-4). An IgD-binding outer membrane protein from H. influenzae (protein D) was isolated and cloned, and shown to be an important pathogenicity factor (5). However, protein D does not bind to the majority of IgD myelomas tested, and it was suggested that encapsulated H. influenzae of serotype b expresses an additional IgD receptor (6). Early studies demonstrated that the outer membrane proteins (OMPs) from a diverse collection of Moraxella isolates exhibit a high degree of similarity (7). Investigators have primarily focused their research efforts on a selected group of proteins. Recent studies have demonstrated that the high-molecular-weight surface antigen, termed UspA or HMW-OMP, is actually comprised of two different proteins. These proteins are named UspA1 and UspA2 (8,9,10). The apparent molecular masses of these OMPs are greater than 250 kDa as determined by SDS-PAGE analysis. Reduction with formic acid yields bands of approximately 120 to 140 kDa, suggesting that the UspA proteins form an oligomeric complex composed of several monomeric subunits (11). The predicted mass of each protein, as deduced from the cloned genes, is 88 kDa and 62 kDa for UspA1 and UspA2, respectively. It is thought that the difference in the deduced mass and the mass determined using SDS-PAGE is due to a predicted coiled coil structure (9). In a recent patent publication, an outer membrane protein of M. catarrhalis with a molecular mass of approximately 200 kDa was isolated (12). A sequence encoding a protein of approximately 200 kDa was also provided. The protein was shown to be immunogenic, but no further biological functions were presented. In addition, a 200 kDa protein is associated with hemagglutinating M. catarrhalis (13,14). CopB is an 80 kDa surface exposed major OMP that shows a moderate antigenic conservation. In addition, OMP CD is a 46 kDa highly conserved protein with numerous surface exposed epitopes and OMP E a 47 kDa protein detected on a variety of heterologous strains. The lactoferrin-binding (LbpA and B) and transferrin-binding (TbpA and B) proteins have molecular sizes of 99-111 and 74-105 kDa, respectively. Certain strains of Staphylococcus aureus produce immunostimulatory exotoxins such as toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin A (SEA), SEB and SEC, all of which are associated with food poisoning and toxic shock syndrome (TSS). These exotoxins have been denominated as superantigens (SAg) due to their ability to activate a high frequency of T lymphocytes. SAg bind as unprocessed proteins to HLA class II molecules on APC and oligoclonally activate T cells expressing particular TCR VD chains. In vivo exposure to excessive amounts of SAg results in a strong cytokine production and includes IL-2, TNF-α and IFN-γ, which are associated with a toxic shock like syndrome. Since the discovery of the first immunoglobulin-binding bacterial protein, S. aureus protein A (SpA) in 1966, this protein has been extensively characterized. The ability of SpA to bind the Fc part of IgG is well known, but SpA also binds a fraction of Ig-molecules of all classes due to the so called ‘alternative’ binding, which represents an interaction with the variable region of certain heavy chains. All IgG-binding capacity of S. aureus has been considered to be mediated by SpA. However, the existence of a second gene in S. aureus encoding an Ig-binding protein has also been reported. Streptococcus pyogenes and Peptostreptococcus magnus are other examples of Ig-binding bacteria. S. pyogenes produces protein H belonging to the M family of proteins, and has strong affinity for the Fc region of IgG. Proteins expressed by some strains bind IgA instead of IgG or both IgG and IgA. Protein Bac or the B-antigen is an IgA-binding protein expressed by certain strains of group B streptococci. Finally, P. magnus expresses protein L that shows high and specific affinity for Ig light chains, especially k light chains, and thereby interacts with all classes of Ig. IgD is a unique immunoglobulin that exists in both a soluble and a surface-bound form. Both forms are encoded by the same gene and are splicing products. All mature B lymphocytes have B cell receptors (BCR) consisting of membrane-bound IgD and IgM. Soluble IgD comprises approximtely 0.25% of the total amount of serum-Ig. The main function of IgD seems to be as an antigen-receptor on the B cell surface in order to optimize B cell recruitment and accelerated affinity maturation. Antigen is taken up through IgD by endocytosis followed by intracellular degradation and presentation on MHC class II for T cells, which in turn are activated and produce cytokines. Hereby, T cell help is obtained including numerous cytokines (e.g. interleukin-4) and co-stimulatory molecules such as CD28. Despite macrophages, dendritic cells, and B cells all can present antigens to T lymphocytes, the B cells are 100-fold more efficient due to the importance of the antigen-presenting immunoglobulin on the surface. An attractive strategy in order to potentiate immunization is to directly target an antigen to the B cell receptor. It was early shown that the mouse antibody-response against bovine serum albumin (BSA) conjugated to anti-IgD monoclonal antibodies was 100-fold stronger compared to BSA administration without any antibody. In paralell, it has been demonstrated that a mouse myeloma antigen incorporated into the constant region of anti-IgD-antibodies targeted to the surface-bound IgD results in an up to 1000-fold more efficient antigen presentation on MHC class II (15). Tolerance induction can be achieved experimentally by B cell activation through the IgD BCR without any additional T cell help. It would also be possible to treat autoimmune diseases by inducing B cell anergy and thus inhibit the production of auto-antibodies. In fact, SLE-prone mice administered dextran-conjugated anti-IgD antibodies exhibit a delayed development of autoimmunity. In yet another study it was shown that B cell activation via IgD decreases a T helper 2-induced IgE response suggesting a therapy for diminishing the IgE production in severely allergic individuals by displacing the antibody response from a Th2- to a Th1-response. By targeting antigens to the B cell receptor IgD, stimulation, tolerance, and a switch from IgE-production can be achieved. In addition, polyclonal activation has been reported. The outcome is depending on the experimental model used. With different constructs including various repeating IgD-binding segments, it is possible to tailor the response. The T cell is a significant player in the anti-tumour response since it recognizes tumour-specific antigens. However, the important T cells display commonly depressed activity in the cancer patient due to a general immunosuppression. A triggering of T helper cells would therefore be very beneficial. Vaccination against tumours using antigen presenting cells (APC) has recently been acknowledged (17). Immunization protocols with APC pulsed ex vivo with tumor antigens (peptides) have been shown to induce effective MHC class I presentation for cytotoxic T cells. It has also been demonstrated that EBV-transformed B cells are able to present melanoma antigens for tumour-infiltrating lymphocytes (TIL). In experimental models, it has also been shown that tumour cells transfected with MHC class II and B7 surface molecules, receptors that are aboundant on B cells, would be a feasible approach for tumour vaccination. Interestingly, B16 melanoma bearing mice that were injected with B cells pulsed with a tumour lysate from the corresponding cell line showed a prolonged survival due to an increase in IFN-γ producing T cells. It was also demonstrated that the induced T helper cells evoked a stronger cytotoxic response against the solid tumours. Since myeloma antigen targeted to IgD induces a T cell response, the suggested approach using IgD-binding bacterial proteins conjugated to specific tumour antigens would be feasible. To target an antigen (e.g. peptide derived from a microbe or a specific tumour) to IgD-bearing B cells in order to trigger both humoral and cellular immune responses a IgD-binding protein or a shorter IgD-binding peptide would be a very feasible vector. Several examples of successful strategies with a similar angle of approach exist. The humoral immune response in mice against bovine serum albumin (BSA) conjugated to anti-IgD monoclonal antibodies is 100-fold stronger compared to when BSA is administered alone. A recent publication by Lunde et al. (15) describes that when a myeloma-derived peptide is integrated in the constant region of anti-IgD Fab′ fragments and injected into mice, a 1,000-fold more efficient antigen presentation is achieved against the antigen in question (15). In parallel, the Ig-binding fragment of S. aureus protein A fused with cholera toxin significantly increases both systemic and mucosal immune responses 10- to 100-fold against the cholera toxin (16). Finally, in a mouse tumour model consisting of the experimentally well defined B16 melanoma, activated B lymphocytes that are pulsed ex vivo with peptides derived from the tumour tissue can evoke a stronger anti-tumour response in vivo and consequently a prolonged survival (17). |
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