Patent Publication Number: US-2004053840-A1

Title: Regulation of human cyclophilin-like protein

Description:
TECHNICAL FIELD OF THE INVENTION  
       [0001] The invention relates to the regulation of human cyclophilin-like protein.  
       BACKGROUND OF THE INVENTION  
       [0002] Cyclophilin accelerates cis-trans peptidyl-prolyl isomerization, which is a slow step in protein folding and unfolding. Ke et al., Proc Natl Acad Sci USA Apr. 15, 1993; 90(8):3324-8; Dorfman et al., J Virol. September 1997; 71(9):7110-3. Additionally, cyclophilin binds the immunosuppressive drug, cyclosporin A (CsA) and is involved in the early lifecycle of HIV. Although the enzymatic and immunosuppressive effects of cyclophilin have been known for some time the actual cellular function of this protein is less well understood. Dolinski et al, Mol Biol Cell. November 1997;8(11):2267-80. Some studies suggest that cyclophilin plays an essential role in protein folding for such proteins as retinal opsins. Studies suggest that cyclophilin may serve an important role in developing cancer specific immunotherapies. There is, therefore, a need in the art to identify additional cyclophilin-like proteins that can be regulated to provide therapeutic effects.  
       SUMMARY OF THE INVENTION  
       [0003] It is an object of the invention to provide reagents and methods of regulating a human cyclophilin-like protein. This and other objects of the invention are provided by one or more of the embodiments described below.  
       [0004] One embodiment of the invention is a cyclophilin-like protein polypeptide comprising an amino acid sequence selected from the group consisting of:  
       [0005] amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2; and  
       [0006] the amino acid sequence shown in SEQ ID NO: 2.  
       [0007] Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a cyclophilin-like protein polypeptide comprising an amino acid sequence selected from the group consisting of:  
       [0008] amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2; and  
       [0009] the amino acid sequence shown in SEQ ID NO: 2.  
       [0010] Binding between the test compound and the cyclophilin-like protein polypeptide is detected. A test compound which binds to the cyclophilin-like protein polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the activity of the cyclophilin-like protein.  
       [0011] Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a polynucleotide encoding a cyclophilin-like protein polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.  
       [0012] Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the cyclophilin-like protein through interacting with the cyclophilin-like protein mRNA.  
       [0013] Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a cyclophilin-like protein polypeptide comprising an amino acid sequence selected from the group consisting of:  
       [0014] amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2; and  
       [0015] the amino acid sequence shown in SEQ ID NO: 2.  
       [0016] A cyclophilin-like protein activity of the polypeptide is detected. A test compound which increases cyclophilin-like protein activity of the polypeptide relative to cyclophilin-like protein activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation. A test compound which decreases cyclophilin-like protein activity of the polypeptide relative to cyclophilin-like protein activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.  
       [0017] Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a cyclophilin-like protein product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:  
       [0018] nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and  
       [0019] the nucleotide sequence shown in SEQ ID NO: 1.  
       [0020] Binding of the test compound to the cyclophilin-like protein product is detected. A test compound which binds to the cyclophilin-like protein product is thereby identified as a potential agent for decreasing extracellular matrix degradation.  
       [0021] Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a cyclophilin-like protein polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:  
       [0022] nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and  
       [0023] the nucleotide sequence shown in SEQ ID NO: 1.  
       [0024] Cyclophilin-like protein activity in the cell is thereby decreased.  
       [0025] The invention thus provides a human cyclophilin-like protein that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme&#39;s active site. Human cyclophilin-like protein and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0026]FIG. 1 shows the DNA-sequence encoding a cyclophilin-like protein Polypeptide (SEQ ID NO:1).  
     [0027]FIG. 2 shows the amino acid sequence deduced from the DNA-sequence of FIG. 1 (SEQ ID NO:2).  
     [0028]FIG. 3 shows the amino acid sequence of the protein identified by SwissProt Accession No. P05092 (SEQ ID NO:3).  
     [0029]FIG. 4 shows the amino acid sequence of a cyclophilin-like protein Polypeptide (SEQ ID NO:4).  
     [0030]FIG. 5 shows the BLASTP—alignment of 299 (SEQ ID NO:2) against swiss|P05092 (SEQ ID NO:3).  
     [0031]FIG. 6 shows the HMMPFAM—alignment of 299 (SEQ ID NO:2) against pfam|hmm|pro_isomerase Cyclophilin-like protein type peptidyl-prolyl cis-tr.  
     [0032]FIG. 7 shows the Promoter predictions for SEQ ID NO:1: 1022 bp upstream from start sequence.  
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0033] The invention relates to an isolated polynucleotide being selected from the group consisting of:  
     [0034] a) a polynucleotide encoding a cyclophilin-like protein polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 88% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.  
     [0035] b) a polynucleotide comprising the sequence of SEQ ID NO: 1;  
     [0036] c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a cyclophilin-like protein polypeptide;  
     [0037] d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a cyclophilin-like protein polypeptide; and  
     [0038] e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a cyclophilin-like protein polypeptide.  
     [0039] Furthermore, it has been discovered by the present applicant that a novel cyclophilin-like protein, particularly a human cyclophilin-like protein, can be used in therapeutic methods to treat cancer. Human cyclophilin-like protein comprises the amino acid sequence shown in SEQ ID NO:2. A coding sequence for human cyclophilin-like protein is shown in SEQ ID NO:1  
     [0040] Human cyclophilin-like protein is 87% identical over 163 amino acids to swiss|P05092 (SEQ ID NO:3). Based on sequence length similarity with human cyclophilin-like protein A, SEQ ID NO:2 represents a full-length sequence. Output from BLAST searches against the protein/DNA sequence databases indicated, by clear homology, that SEQ ID NO:2 encodes a cyclophilin-like protein (FIG. 5). A search against protein motif databases with SEQ ID NO:2 identified cyclophilin-like protein type peptidyl-prolyl cis-tr isomerase region, and the 3D structure of SEQ ID NO:2 is inferred by clear sequence similarity to a cyclophilin-like protein (FIG. 6).  
     [0041] The consensus pattern, [FY]-x(2)-[STCNLV]-x-F-H-[RH]-[LIVMN]-[LIVM]-x(2)-F-[LIVM]-x-Q-[AG]-G, identified in PROSITE is found in SEQ ID NO:2 except for the substitution Q62H. This glutamine to histidine substitution in the consensus pattern may not necessarily abolish the functionality of SEQ ID NO:2. First, the Phe, Met, Phe, Leu, and His residues (underlined in FIG. 5), which form the hydrophobic pocket and are critical for substrate binding, remain in SEQ ID NO:2. Moreover, according to the 3-d structure model, the Gln62 lies in a beta strand away from the critical residues underlined in FIG. 5. Additionally, the consensus sequence described in Prosite does not identify all Cyclophilin-like protein-type peptidyl-prolyl cis-trans isomerases. Seven known cyclophilin-like protein-type peptidyl-prolyl cis-trans isomerase were not identified by the consensus sequence. For example, a peptidyl-prolyl cis-trans isomerase (Accession No. Q50639; SEQ ID NO:4) from  Mycobacterium tuberculosis  does not have the consensus sequence above.  
     [0042] Human cyclophilin-like protein of the invention is expected to be useful for the same purposes as previously identified cyclophilin-like protein. Human cyclophilin-like protein is believed to be useful in therapeutic methods to treat disorders such as cancer. Human cyclophilin-like protein also can be used to screen for human cyclophilin-like protein activators and inhibitors.  
     [0043] Polypeptides  
     [0044] Human cyclophilin-like protein polypeptides according to the invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, or 164 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof, as defined below. A cyclophilin-like protein polypeptide of the invention therefore can be a portion of a cyclophilin-like protein, a full-length cyclophilin-like protein, or a fusion protein comprising all or a portion of a cyclophilin-like protein.  
     [0045] Biologically Active Variants  
     [0046] Human cyclophilin-like protein polypeptide variants that are biologically active, e.g., retain a prolyl isomerase activity, also are cyclophilin-like protein polypeptides. Preferably, naturally or non-naturally occurring cyclophilin-like protein polypeptide variants have amino acid sequences which are at least about 88, 90, 96, 96, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO:2 or a fragment thereof. Percent identity between a putative cyclophilin-like protein polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).  
     [0047] Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.  
     [0048] Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a cyclophilin-like protein polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active cyclophilin-like protein polypeptide can readily be determined by assaying for prolyl isomerase activity, as described for example, in Dolinski et al., supra.  
     [0049] Fusion Proteins  
     [0050] Fusion proteins are useful for generating antibodies against cyclophilin-like protein polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a cyclophihn-like protein polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.  
     [0051] A cyclophilin-like protein polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, or 164 contiguous amino acids of SEQ ID NO:2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length cyclophilin-like protein.  
     [0052] The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the cyclophilin-like protein polypeptide-encoding sequence and the heterologous protein sequence, so that the cyclophilin-like protein polypeptide can be cleaved and purified away from the heterologous moiety.  
     [0053] A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from the complement of SEQ ID NO:1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).  
     [0054] Identification of Species Homologs  
     [0055] Species homologs of human cyclophilin-like protein polypeptide can be obtained using cyclophilin-like protein polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of cyclophilin-like protein polypeptide, and expressing the cDNAs as is known in the art.  
     [0056] Polynucleotides  
     [0057] A cyclophilin-like protein polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a cyclophilin-like protein polypeptide. A coding sequence for human cyclophilin-like protein is shown in SEQ ID NO:1; this sequence is located on chromosome 2.  
     [0058] Degenerate nucleotide sequences encoding human cyclophilin-like protein polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO:1 or its complement also are cyclophilin-like protein polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of −12 and a gap extension penalty of −2. Complementary DNA (cDNA) molecules, species homologs, and variants of cyclophilin-like protein polynucleotides that encode biologically active cyclophilin-like protein polypeptides also are cyclophilin-like protein polynucleotides. Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO:1 or its complement also are cyclophilin-like protein polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.  
     [0059] Identification of Polynucleotide Variants and Homologs  
     [0060] Variants and homologs of the cyclophilin-like protein polynucleotides described above also are cyclophilin-like protein polynucleotides. Typically, homologous cyclophilin-like protein polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known cyclophilin-like protein polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions—2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.  
     [0061] Species homologs of the cyclophilin-like protein polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of cyclophilin-like protein polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m  of a double-stranded DNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al.,  J. Mol. Biol.  81, 123 (1973). Variants of human cyclophilin-like protein polynucleotides or cyclophilin-like protein polynucleotides of other species can therefore be identified by hybridizing a putative homologous cyclophilin-like protein polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:1 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.  
     [0062] Nucleotide sequences which hybridize to cyclophilin-like protein polynucleotides or their complements following stringent hybridization and/or wash conditions also are cyclophilin-like protein polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., M OLECULAR  C LONING:  A L ABORATORY  M ANUAL,  2d ed., 1989, at pages 9.50-9.51.  
     [0063] Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20° C. below the calculated T m  of the hybrid under study. The T m  of a hybrid between a cyclophilin-like protein polynucleotide having a nucleotide sequence shown in SEQ ID NO:1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy,  Proc. Natl. Acad. Sci. U.S.A.  48, 1390 (1962): 
       T   m =81.5° C.−16.6(log 10 [Na + ])+0.41(% G+C )−0.63(%formamide)−600/l), 
     [0064] where l=the length of the hybrid in basepairs.  
     [0065] Stringent wash conditions include, for example, 4×SSC at 65° C., or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highly stringent wash conditions include, for example, 0.2×SSC at 65° C.  
     [0066] Preparation of Polynucleotides  
     [0067] A cyclophilin-like protein polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated cyclophilin-like protein polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises cyclophilin-like protein nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.  
     [0068] Human cyclophilin-like protein cDNA molecules can be made with standard molecular biology techniques, using cyclophilin-like protein mRNA as a template. Human cyclophilin-like protein cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.  
     [0069] Alternatively, synthetic chemistry techniques can be used to synthesize cyclophilin-like protein polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a cyclophilin-like protein polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof.  
     [0070] Extending Polynucleofides  
     [0071] Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar,  PCR Methods Applic.  2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.  
     [0072] Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al.,  Nucleic Acids Res.  16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.  
     [0073] Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al.,  PCR Methods Applic.  1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.  
     [0074] Another method which can be used to retrieve unknown sequences is that of Parker et al.,  Nucleic Acids Res.  19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.  
     [0075] When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5′ regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5′ non-transcribed regulatory regions.  
     [0076] Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.  
     [0077] Obtaining Polypeptides  
     [0078] Human cyclophilin-like protein polypeptides can be obtained, for example, by purification from human cells, by expression of cyclophilin-like protein polynucleotides, or by direct chemical synthesis.  
     [0079] Protein Purification  
     [0080] Human cyclophilin-like protein polypeptides can be purified from any cell that expresses the enzyme, including host cells that have been transfected with cyclophilin-like protein expression constructs. A purified cyclophilin-like protein polypeptide is separated from other compounds that normally associate with the cyclophilin-like protein polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified cyclophilin-like protein polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.  
     [0081] Expression of Polynucleotides  
     [0082] To express a cyclophilin-like protein polynucleotide, the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding cyclophilin-like protein polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., C URRENT  P ROTOCOLS IN  M OLECULAR  B IOLOGY,  John Wiley &amp; Sons, New York, N.Y., 1989.  
     [0083] A variety of expression vector/host systems can be utilized to contain and express sequences encoding a cyclophilin-like protein polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.  
     [0084] The control elements or regulatory sequences are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a cyclophilin-like protein polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.  
     [0085] Bacterial and Yeast Expression Systems  
     [0086] In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the cyclophilin-like protein polypeptide. For example, when a large quantity of a cyclophilin-like protein polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional  E. coli  cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding the cyclophilin-like protein polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke &amp; Schuster,  J. Biol. Chem.  264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.  
     [0087] In the yeast  Saccharomyces cerevisiae,  a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al.,  Methods Enzymol.  153, 516-544, 1987.  
     [0088] Plant and Insect Expression Systems  
     [0089] If plant expression vectors are used, the expression of sequences encoding cyclophilin-like protein polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu,  EMBO J.  6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al.,  EMBO J.  3, 1671-1680, 1984; Broglie et al.,  Science  224, 838-843, 1984; Winter et al., Results  Probl. Cell Differ.  17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in M C G RAW  H ILL  Y EARBOOK OF  S CIENCE AND  T ECHNOLOGY,  McGraw Hill, New York, N.Y., pp. 191-196, 1992).  
     [0090] An insect system also can be used to express a cyclophilin-like protein polypeptide. For example, in one such system  Autographa californica  nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in  Spodoptera frugiperda  cells or in Trichoplusia larvae. Sequences encoding cyclophilin-like protein polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of cyclophilin-like protein polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect  S. frugiperda  cells or Trichoplusia larvae in which cyclophilin-like protein polypeptides can be expressed (Engelhard et al.,  Proc. Nat. Acad. Sci.  91, 3224-3227, 1994).  
     [0091] Mammalian Expression Systems  
     [0092] A number of viral-based expression systems can be used to express cyclophilin-like protein polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding cyclophilin-like protein polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a cyclophilin-like protein polypeptide in infected host cells (Logan &amp; Shenk,  Proc. Natl. Acad. Sci.  81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.  
     [0093] Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).  
     [0094] Specific initiation signals also can be used to achieve more efficient translation of sequences encoding cyclophilin-like protein polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a cyclophilin-like protein polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al.,  Results Probl. Cell Differ.  20, 125-162, 1994).  
     [0095] Host Cells  
     [0096] A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed cyclophilin-like protein polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.  
     [0097] Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express cyclophilin-like protein polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced cyclophilin-like protein sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, A NIMAL  C ELL  C ULTURE,  R. I. Freshney, ed., 1986.  
     [0098] Any number of selection systems can be used to recover transformed cell lines.  
     [0099] These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al.,  Cell  11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al.,  Cell  22, 817-23, 1980) genes which can be employed in tk −  or aprt −  cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al.,  Proc. Natl. Acad. Sci.  77, 3567-70, 1980), npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al.,  J. Mol. Biol.  150, 1-14, 1981), and als and pat confer resistance to chlorsulfiron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman &amp; Mulligan,  Proc. Natl. Acad. Sci.  85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al.,  Methods Mol. Biol.  55, 121-131, 1995).  
     [0100] Detecting Expression  
     [0101] Although the presence of marker gene expression suggests that the cyclophilin-like protein polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a cyclophilin-like protein polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a cyclophilin-like protein polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a cyclophilin-like protein polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the cyclophilin-like protein polynucleotide.  
     [0102] Alternatively, host cells which contain a cyclophilin-like protein polynucleotide and which express a cyclophilin-like protein polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a cyclophilin-like protein polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a cyclophilin-like protein polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a cyclophilin-like protein polypeptide to detect transformants that contain a cyclophilin-like protein polynucleotide.  
     [0103] A variety of protocols for detecting and measuring the expression of a cyclophilin-like protein polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a cyclophilin-like protein polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al., S EROLOGICAL  M ETHODS : A L ABORATORY  M ANUAL,  APS Press, St. Paul, Minn., 1990) and Maddox et al.,  J. Exp. Med.  158, 1211-1216, 1983).  
     [0104] A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding cyclophilin-like protein polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a cyclophilin-like protein polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.  
     [0105] Expression and Purification of Polypeptides  
     [0106] Host cells transformed with nucleotide sequences encoding a cyclophilin-like protein polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode cyclophilin-like protein polypeptides can be designed to contain signal sequences which direct secretion of soluble cyclophilin-like protein polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound cyclophilin-like protein polypeptide.  
     [0107] As discussed above, other constructions can be used to join a sequence encoding a cyclophilin-like protein polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the cyclophilin-like protein polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a cyclophilin-like protein polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al.,  Prot. Exp. Purif.  3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the cyclophilin-like protein polypeptide from the fusion protein. Vectors that contain fusion proteins are disclosed in Kroll et al.,  DNA Cell Biol.  12, 441-453, 1993.  
     [0108] Chemical Synthesis  
     [0109] Sequences encoding a cyclophilin-like protein polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al.,  Nucl. Acids Res. Symp. Ser.  215-223, 1980; Horn et al.  Nucl. Acids Res. Symp. Ser.  225-232, 1980). Alternatively, a cyclophilin-like protein polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield,  J. Am. Chem. Soc.  85, 2149-2154, 1963; Roberge et al.,  Science  269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of cyclophilin-like protein polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.  
     [0110] The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, P ROTEINS:  S TRUCTURES AND  M OLECULAR  P RINCIPLES,  W H Freeman and Co., New York, N.Y., 1983). The composition of a synthetic cyclophilin-like protein polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the cyclophilin-like protein polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.  
     [0111] Production of Altered Polypeptides  
     [0112] As will be understood by those of skill in the art, it may be advantageous to produce cyclophilin-like protein polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.  
     [0113] The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter cyclophilin-like protein polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.  
     [0114] Antibodies  
     [0115] Any type of antibody known in the art can be generated to bind specifically to an epitope of a cyclophilin-like protein polypeptide. “Antibody” as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab′) 2 , and Fv, which are capable of binding an epitope of a cyclophilin-like protein polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.  
     [0116] An antibody which specifically binds to an epitope of a cyclophilin-like protein polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.  
     [0117] Typically, an antibody which specifically binds to a cyclophilin-like protein polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to cyclophilin-like protein polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a cyclophilin-like protein polypeptide from solution.  
     [0118] Human cyclophilin-like protein polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a cyclophilin-like protein polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund&#39;s adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and  Corynebacterium parvum  are especially useful.  
     [0119] Monoclonal antibodies that specifically bind to a cyclophilin-like protein polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al.,  Nature  256, 495-497, 1985; Kozbor et al.,  J. Immunol. Methods  81, 31-42, 1985; Cote et al.,  Proc. Natl. Acad. Sci.  80, 2026-2030, 1983; Cole et al.,  Mol. Cell Biol.  62, 109-120, 1984).  
     [0120] In addition, techniques developed for the production of “chimeric antibodies,” the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al.,  Proc. Natl. Acad. Sci.  81, 6851-6855, 1984; Neuberger et al.,  Nature  312, 604-608, 1984; Takeda et al.,  Nature  314, 452454, 1985). Monoclonal and other antibodies also can be “humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies that specifically bind to a cyclophilin-like protein polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.  
     [0121] Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to cyclophilin-like protein polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton,  Proc. Natl. Acad. Sci.  88, 11120-23, 1991).  
     [0122] Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., 1996,  Eur. J. Cancer Prev.  5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma &amp; Morrison, 1997,  Nat. Biotechnol.  15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender &amp; Voss, 1994,  J. Biol. Chem.  269, 199-206.  
     [0123] A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., 1995,  Int. J. Cancer  61, 497-501; Nicholls et al., 1993,  J. Immunol. Meth.  165, 81-91).  
     [0124] Antibodies which specifically bind to cyclophilin-like protein polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al.,  Proc. Natl. Acad. Sci.  86, 3833-3837, 1989; Winter et al.,  Nature  349, 293-299, 1991).  
     [0125] Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the “diabodies” described in WO 94/13804, also can be prepared.  
     [0126] Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a cyclophilin-like protein polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.  
     [0127] Antisense Oligonucleotides  
     [0128] Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of cyclophilin-like protein gene products in the cell.  
     [0129] Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5′ end of one nucleotide with the 3′ end of another nucleotide with non-phosphodiester intemucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown,  Meth. Mol. Biol.  20, 1-8, 1994; Sonveaux,  Meth. Mol. Biol.  26, 1-72, 1994; Uhlmann et al.,  Chem. Rev.  90, 543-583, 1990.  
     [0130] Modifications of cyclophilin-like protein gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5′, or regulatory regions of the cyclophilin-like protein gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using “triple helix” base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al., in Huber &amp; Carr, M OLECULAR AND  I MMUNOLOGIC  A PPROACHES,  Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.  
     [0131] Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a cyclophilin-like protein polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a cyclophilin-like protein polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent cyclophilin-like protein nucleotides, can provide sufficient targeting specificity for cyclophilin-like protein mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular cyclophilin-like protein polynucleotide sequence.  
     [0132] Antisense oligonucleotides can be modified without affecting their ability to hybridize to a cyclophilin-like protein polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the 5′ phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al.,  Trends Biotechnol.  10, 152-158, 1992; Uhlmann et al.,  Chem. Rev.  90, 543-584, 1990; Uhlmann et al.,  Tetrahedron. Lett.  215, 3539-3542, 1987.  
     [0133] Ribozymes  
     [0134] Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech,  Science  236, 1532-1539; 1987; Cech,  Ann. Rev. Biochem.  59, 543-568; 1990, Cech,  Curr. Opin. Struct. Biol.  2, 605-609; 1992, Couture &amp; Stinchcomb,  Trends Genet  12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.  
     [0135] The coding sequence of a cyclophilin-like protein polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the cyclophilin-like protein polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al.  Nature  334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete “hybridization” region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201).  
     [0136] Specific ribozyme cleavage sites within a cyclophilin-like protein RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate cyclophilin-like protein RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.  
     [0137] Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease cyclophilin-like protein expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.  
     [0138] As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.  
     [0139] Differentially Expressed Genes  
     [0140] Described herein are methods for the identification of genes whose products interact with human cyclophilin-like. protein. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, cancer. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human cyclophilin-like protein gene or gene product may itself be tested for differential expression.  
     [0141] The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.  
     [0142] Identification of Differentially Expressed Genes  
     [0143] To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al., ed., C URRENT  P ROTOCOLS IN  M OLECULAR  B IOLOGY,  John Wiley &amp; Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Pat. No. 4,843,155.  
     [0144] Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al.,  Proc. Natl. Acad. Sci. U.S.A.  85, 208-12, 1988), subtractive hybridization (Hedrick et al.,  Nature  308, 149-53; Lee et al.,  Proc. Natl. Acad. Sci. U.S.A.  88, 2825, 1984), and, preferably, differential display (Liang &amp; Pardee,  Science  257, 967-71, 1992; U.S. Pat. No. 5,262,311).  
     [0145] The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human cyclophilin-like protein. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human cyclophilin-like protein. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human cyclophilin-like protein gene or gene product are up-regulated or down-regulated.  
     [0146] Screening Methods  
     [0147] The invention provides assays for screening test compounds that bind to or modulate the activity of a cyclophilin-like protein polypeptide or a cyclophilin-like protein polynucleotide. A test compound preferably binds to a cyclophilin-like protein polypeptide or polynucleotide. More preferably, a test compound decreases or increases prolyl isomerase activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.  
     [0148] Test Compounds  
     [0149] Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam,  Anticancer Drug Des.  12, 145, 1997.  
     [0150] Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al.,  Proc. Natl. Acad. Sci. U.S.A.  90, 6909, 1993; Erb et al.  Proc. Natl. Acad. Sci. U.S.A.  91, 11422, 1994; Zuckermann et al.,  J. Med. Chem.  37,2678, 1994; Cho et al.,  Science  261, 1303, 1993; Carell et al.,  Angew. Chem. Int. Ed. Engl.  33, 2059, 1994; Carell et al.,  Angew. Chem. Int. Ed. Engl.  33, 2061; Gallop et al.,  J. Med. Chem.  37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten,  BioTechniques  13, 412-421, 1992), or on beads (Lam,  Nature  354, 82-84, 1991), chips (Fodor,  Nature  364, 555-556, 1993), bacteria or spores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al.,  Proc. Natl. Acad. Sci. U.S.A.  89, 1865-1869, 1992), or phage (Scott &amp; Smith,  Science  249, 386-390, 1990; Devlin,  Science  249, 404-406, 1990); Cwirla et al.,  Proc. Natl. Acad. Sci.  97, 6378-6382, 1990; Felici,  J. Mol. Biol.  222, 301-310, 1991; and Ladner, U.S. Pat. No. 5,223,409).  
     [0151] High Throughput Screening  
     [0152] Test compounds can be screened for the ability to bind to cyclophilin-like protein polypeptides or polynucleotides or to affect cyclophilin-like protein activity or cyclophilin-like protein gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.  
     [0153] Alternatively, “free format assays,” or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al.,  Proc. Natl. Acad. Sci. U.S.A.  19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.  
     [0154] Another example of a free format assay is described by Chelsky, “Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches,” reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.  
     [0155] Yet another example is described by Salmon et al.,  Molecular Diversity  2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.  
     [0156] Another high throughput screening method is described in Beutel et al., U.S. Pat. No. 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.  
     [0157] Binding Assays  
     [0158] For binding assays, the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the cyclophilin-like protein polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.  
     [0159] In binding assays, either the test compound or the cyclophilin-like protein polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the cyclophilin-like protein polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.  
     [0160] Alternatively, binding of a test compound to a cyclophilin-like protein polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a cyclophilin-like protein polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a cyclophilin-like protein polypeptide (McConnell et al.,  Science  257, 1906-1912, 1992).  
     [0161] Determining the ability of a test compound to bind to a cyclophilin-like protein polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander &amp; Urbaniczky,  Anal. Chem.  63, 2338-2345, 1991, and Szabo et al.,  Curr. Opin. Struct. Biol.  5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.  
     [0162] In yet another aspect of the invention, a cyclophilin-like protein polypeptide can be used as a “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.,  Cell  72, 223-232, 1993; Madura et al.,  J. Biol. Chem.  268, 12046-12054, 1993; Bartel et al.,  BioTechniques  14, 920-924, 1993; Iwabuchi et al.,  Oncogene  8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the cyclophilin-like protein polypeptide and modulate its activity.  
     [0163] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding a cyclophilin-like protein polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein (“prey” or “sample”) can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the cyclophilin-like protein polypeptide.  
     [0164] It may be desirable to immobilize either the cyclophilin-like protein polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the cyclophilin-like protein polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a cyclophilin-like protein polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants.  
     [0165] Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.  
     [0166] In one embodiment, the cyclophilin-like protein polypeptide is a fusion protein comprising a domain that allows the cyclophilin-like protein polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed cyclophilin-like protein polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.  
     [0167] Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a cyclophilin-like protein polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated cyclophilin-like protein polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a cyclophilin-like protein polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the cyclophilin-like protein polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.  
     [0168] Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the cyclophilin-like protein polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the cyclophilin-like protein polypeptide, and SDS gel electrophoresis under non-reducing conditions.  
     [0169] Screening for test compounds which bind to a cyclophilin-like protein polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a cyclophilin-like protein polypeptide or polynucleotide can be used in a cell-based assay system. A cyclophilin-like protein polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a cyclophilin-like protein polypeptide or polynucleotide is determined as described above.  
     [0170] Functional Assays  
     [0171] Test compounds can be tested for the ability to increase or decrease the prolyl isomerase activity of a human cyclophilin-like protein polypeptide. Prolyl isomerase activity can be measured, for example, as described in Dolinski et al., supra.  
     [0172] Functional assays can be carried out after contacting either a purified cyclophilin-like protein polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases a prolyl isomerase activity of a cyclophilin-like protein polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing cyclophilin-like protein activity. A test compound which increases a prolyl isomerase activity of a human cyclophilin-like protein polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human cyclophilin-like protein activity.  
     [0173] Gene Expression  
     [0174] In another embodiment, test compounds that increase or decrease cyclophilin-like protein gene expression are identified. A cyclophilin-like protein polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the cyclophilin-like protein polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.  
     [0175] The level of cyclophilin-like protein mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a cyclophilin-like protein polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a cyclophilin-like protein polypeptide.  
     [0176] Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a cyclophilin-like protein polynucleotide can be used in a cell-based assay system. The cyclophilin-like protein polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.  
     [0177] Pharmaceutical Compositions  
     [0178] The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a cyclophilin-like protein polypeptide, cyclophilin-like protein polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a cyclophilin-like protein polypeptide, or mimetics, activators, or inhibitors of a cyclophilin-like protein polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.  
     [0179] In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous; intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.  
     [0180] Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.  
     [0181] Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie., dosage.  
     [0182] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.  
     [0183] Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks&#39; solution, Ringer&#39;s solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.  
     [0184] The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.  
     [0185] Further details on techniques for formulation and administration can be found in the latest edition of R EMINGTON&#39;S  P HARMACEUTICAL  S CIENCES  (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.  
     [0186] Therapeutic Indications and Methods  
     [0187] Human cyclophilin-like protein can be regulated to treat cancer. Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.  
     [0188] Most standard cancer therapies target cellular proliferation and rely on the differential proliferative capacities between transformed and normal cells for their efficacy. This approach is hindered by the facts that several important normal cell types are also highly proliferative and that cancer cells frequently become resistant to these agents. Thus, the therapeutic indices for traditional anti-cancer therapies rarely exceed 2.0.  
     [0189] The advent of genomics-driven molecular target identification has opened up the possibility of identifying new cancer-specific targets for therapeutic intervention that will provide safer, more effective treatments for cancer patients. Thus, newly discovered tumor-associated genes and their products can be tested for their role(s) in disease and used as tools to discover and develop innovative therapies. Genes playing important roles in any of the physiological processes outlined above can be characterized as cancer targets.  
     [0190] Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins. These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Agonists and/or antagonists of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.  
     [0191] This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a cyclophilin-like protein polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.  
     [0192] A reagent which affects cyclophilin-like protein activity can be administered to a human cell, either in vitro or in vivo, to reduce cyclophilin-like protein activity. The reagent preferably binds to an expression product of a human cyclophilin-like protein gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.  
     [0193] In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.  
     [0194] A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 10 6  cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10 6  cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10 6  cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.  
     [0195] Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.  
     [0196] Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Pat. No. 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.  
     [0197] In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al.  Trends in Biotechnol.  11, 202-05 (1993); Chiou et al., G ENE  T HERAPEUTICS:  M ETHODS AND  A PPLICATIONS OF  D IRECT  G ENE  T RANSFER  (J. A. Wolff, ed.) (1994); Wu &amp; Wu,  J. Biol. Chem.  263, 621-24 (1988); Wu et al.,  J. Biol. Chem.  269, 542-46 (1994); Zenke et al.,  Proc. Natl. Acad. Sci. U.S.A.  87, 3655-59 (1990); Wu et al.,  J. Biol. Chem.  266, 338-42 (1991).  
     [0198] Determination of a Therapeutically Effective Dose  
     [0199] The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases cyclophilin-like protein activity relative to the cyclophilin-like protein activity which occurs in the absence of the therapeutically effective dose.  
     [0200] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine usefull doses and routes for administration in humans.  
     [0201] Therapeutic efficacy and toxicity, e.g., ED 50  (the dose therapeutically effective in 50% of the population) and LD 50  (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .  
     [0202] Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50  with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.  
     [0203] The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.  
     [0204] Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.  
     [0205] If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular traportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, “gene gun,” and DEAE- or calcium phosphate-mediated transfection.  
     [0206] Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.  
     [0207] If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.  
     [0208] Preferably, a reagent reduces expression of a cyclophilin-like protein gene or the activity of a cyclophilin-like protein polypeptide by at least about 10, preferably about 50, more preferably. about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a cyclophilin-like protein gene or the activity of a cyclophilin-like protein polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to cyclophilin-like protein-specific mRNA, quantitative RT-PCR, immunologic detection of a cyclophilin-like protein polypeptide, or measurement of cyclophilin-like protein activity.  
     [0209] In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.  
     [0210] Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.  
     [0211] Diagnostic Methods  
     [0212] Human cyclophilin-like protein also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding cyclophilin-like protein in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.  
     [0213] Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.  
     [0214] Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al.,  Science  230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al.,  Proc. Natl. Acad. Sci. USA  85, 4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.  
     [0215] Altered levels of a cyclophilin-like protein also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.  
     [0216] All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.  
     EXAMPLE 1  
     [0217] Detection of Cyclophilin-Like Protein Activity  
     [0218] The polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-cyclophilin-like protein polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and peptidyl prolyl cis-trans isomerase activity is measured in an assay containing 50 μl of the cell extract in 50 mM Hepes, 100 mM NaCl, pH 8.0 (875 μl), in a photometer cuvette and cooled to 10° C. Then, 50 μl of alpha-chymotrypsin (Serva, Heidelberg, Germany) (10 mg/ml in 1 mM HCl) are added, and the reaction is started by addition of 25 μl 4 mM N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Bachem, Bubendorf, Switzerland) in trifluorethanol/470 mM lithium chloride. The increase in absorbance at 390 nm is recorded with a Cary 1E spectrophotometer. Data points are obtained every 0.5 s and fitted to a first-order rate law to obtain the reaction rate in units of absorbance (ABS) per min. It is shown that the polypeptide of SEQ ID NO: 2 has a cyclophilin-like protein activity.  
     EXAMPLE 2  
     [0219] Expression of Recombinant Human Cyclophilin-Like Protein  
     [0220] The  Pichia pastoris  expression vector pPICZB (Invitrogen, San Diego, Calif.) is used to produce large quantities of recombinant human cyclophilin-like protein polypeptides in yeast. The cyclophilin-like protein-encoding DNA sequence is derived from SEQ ID NO:1. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5′-end an initiation codon and at its 3′-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in  Pichia pastoris,  driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.  
     [0221] The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, Calif.) according to manufacturer&#39;s instructions. Purified human cyclophilin-like protein polypeptide is obtained.  
     EXAMPLE 3  
     [0222] Identification of Test Compounds that Bind to Cyclophilin-Like Protein Polypeptides  
     [0223] Purified cyclophilin-like protein polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human cyclophilin-like protein polypeptides comprise the amino acid sequence shown in SEQ ID NO:2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.  
     [0224] The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a cyclophilin-like protein polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a cyclophilin-like protein polypeptide.  
     EXAMPLE 4  
     [0225] Identification of a Test Compound which Decreases Cyclophilin-Like Protein Gene Expression  
     [0226] A test compound is administered to a culture of human cells transfected with a cyclophilin-like protein expression construct and incubated at 37° C. for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.  
     [0227] RNA is isolated from the two cultures as described in Chirgwin et al.,  Biochem.  18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a  32 P-labeled cyclophilin-like protein-specific probe at 65° C. in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:1. A test compound that decreases the cyclophilin-like protein-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of cyclophilin-like protein gene expression.  
     EXAMPLE 5  
     [0228] Identification of a Test Compound which Decreases Cyclophilin-Like Protein Activity  
     [0229] A test compound is administered to a culture of human cells transfected with a cyclophilin-like protein expression construct and incubated at 37° C. for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control. cyclophilin-like protein activity is measured using the method of Dolinsi et al., supra.  
     [0230] A test compound which decreases the cyclophilin-like protein activity of the cyclophilin-like protein relative to the cyclophilin-like protein activity in the absence of the test compound is identified as an inhibitor of cyclophilin-like protein activity.  
     EXAMPLE 6  
     [0231] Tissue-Specific Expression of Cyclophilin-Like Protein  
     [0232] The qualitative expression pattern of cyclophilin-like protein in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). To demonstrate that cyclophilin-like protein is involved in cancer, expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes. Expression in the following cancer cell lines also is determined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205 (colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon), MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignant and normal tissue from the same patient also are tested.  
     [0233] Quantitative expression profiling. Quantitative expression profiling is performed by the form of quantitative PCR analysis called “kinetic analysis” firstly described in Higuchi et al.,  BioTechnology  10, 413-17, 1992, and Higuchi et al.,  BioTechnology  11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.  
     [0234] If the amplification is performed in the presence of an internally quenched fluorescent oligonucleotide (TaqMan probe) complementary to the target sequence, the probe is cleaved by the 5′-3′ endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al.,  Proc. Natl. Acad. Sci. U.S.A.  88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al.,  Genome Res.  6, 986-94, 1996, and Gibson et al.,  Genome Res.  6, 995-1001, 1996).  
     [0235] The amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction. In this kind of experiment, the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used.  
     [0236] All “real time PCR” measurements of fluorescence are made in the ABI Prism 7700.  
     [0237] RNA extraction and cDNA preparation. Total RNA from the tissues listed above are used for expression quantification. RNAs labeled “from autopsy” were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer&#39;s protocol.  
     [0238] Fifty μg of each RNA were treated with DNase I for 1 hour at 37° C. in the following reaction mix: 0.2 U/μl RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, Calif.); 10 mM Tris-HCl pH 7.9; 10 mM MgCl 2 ; 50 mM NaCl; and 1 mM DTT.  
     [0239] After incubation, RNA is extracted once with 1 volume of phenol:chloroform:isoamyl alcohol (24:24:1) and once with chloroform, and precipitated with {fraction (1/10)} volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol.  
     [0240] Fifty μg of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, Tex.). After resuspension and spectro-photometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, Calif.) according to the manufacturer&#39;s protocol. The final concentration of RNA in the reaction mix is 200 ng/μL. Reverse transcription is carried out with 2.5 μM of random hexamer primers.  
     [0241] TaqMan quantitative analysis. Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5′ end FAM (6-carboxy-fluorescein) and at the 3′ end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.  
     [0242] Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, Calif.).  
     [0243] The assay reaction mix is as follows: 1× final TaqMan Universal PCR Master Mix (from 2× stock) (PE Applied Biosystems, Calif.); 1X PDAR control—18S RNA (from 20× stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 μl.  
     [0244] Each of the following steps are carried out once: pre PCR, 2 minutes at 50° C., and 10 minutes at 95° C. The following steps are carried out 40 times: denaturation, 15 seconds at 95° C., annealing/extension, 1 minute at 60° C.  
     [0245] The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, Calif.). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user&#39;s manual in order to achieve better background subtraction as well as signal linearity with the staring target quantity.  
     EXAMPLE 7  
     [0246] Proliferation Inhibition Assay: Antisense Oligonucleotides Suppress the Growth of Cancer Cell Lines  
     [0247] The cell line used for testing is the human colon cancer cell line HCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37° C. in a 95% air/5%CO 2  atmosphere.  
     [0248] Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems Model 380B DNA synthesizer using phosphoroamidite chemistry. A sequence of 24 bases complementary to the nucleotides at position 1 to 24 of SEQ ID NO:1 is used as the test oligonucleotide. As a control, another (random) sequence is used: 5′-TCA ACT GAC TAG ATG TAC ATG GAC-3′. Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration. Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 μM once per day for seven days.  
     [0249] The addition of the test oligonucleotide for seven days results in significantly reduced expression of human cyclophilin as determined by Western blotting. This effect is not observed with the control oligonucleotide. After 3 to 7 days, the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide. The number of cells in cultures treated with the test oligonucleotide is not more than 30% of control, indicating that the inhibition of human cyclophilin 1 has an anti-proliferative effect on cancer cells.  
     EXAMPLE 8  
     [0250] In vivo Testing of Compounds/Target Validation  
     [0251] 1. Acute Mechanistic Assays  
     [0252] 1.1. Reduction in Mitogenic Plasma Hormone Levels  
     [0253] This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus. Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.). At a predetermined time after administration of test compound, blood plasma is collected. Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m. into mice at a dosage of 30 ng/mouse to induce a burst of testosterone synthesis). The timing of plasma collection would be adjusted to coincide with the peak of the induced hormone response. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student&#39;s t-test. Significance is p value≦0.05 compared to the vehicle control group.  
     [0254] 1.2. Hollow Fiber Mechanism of Action Assay  
     [0255] Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student&#39;s t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p≦0.05 as compared to the vehicle control group.  
     [0256] 2. Subacute Functional In Vivo Assays  
     [0257] 2.1. Reduction in Mass of Hormone Dependent Tissues  
     [0258] This is another non-tumor assay that measures the ability of a compound to reduce the mass of a hormone dependent tissue (i.e., seminal vesicles in males and uteri in females). Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for a predetermined duration (i.e., 1 week). At termination of the study, animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded. Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent. Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student&#39;s t-test. Significance is p value≦0.05 compared to the vehicle control group.  
     [0259] 2.2. Hollow Fiber Proliferation Assay  
     [0260] Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol. Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student&#39;s t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p≦0.05 as compared to the vehicle control group.  
     [0261] 2.3. Anti-Angiogenesis Models  
     [0262] 2.3.1. Corneal Angiogenesis  
     [0263] Hydron pellets with or without growth factors or cells are implanted into a micropocket surgically created in the rodent cornea. Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet). Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Rank Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student&#39;s t-test (2 tail). Significance is p≦0.05 as compared to the growth factor or cells only group.  
     [0264] 2.3.2. Matrigel Angiogenesis  
     [0265] Matrigel, containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student&#39;s t-test, after the variance between groups is compared by an F-test, with significance determined at p≦0.05 as compared to the vehicle control group.  
     [0266] 3. Primary Antitumor Efficacy  
     [0267] 3.1. Early Therapy Models  
     [0268] 3.1.1. Subcutaneous Tumor  
     [0269] Tumor cells or fragments are implanted subcutaneously on Day 0. Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden. Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day. Anti-tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student&#39;s t-test, after the variance between groups is compared by an F-test, with significance determined at p≦0.05. The experiment may also be continued past the end of dosing in which case tumor measurements would continue to be recorded to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p≦0.05.  
     [0270] 3.1.2. Intraperitoneal/Intracranial Tumor Models  
     [0271] Tumor cells are injected intraperitoneally or intracranially on Day 0. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting on Day 1. Observations of morbidity and/or mortality are recorded twice daily. Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long-term survivors is indicated separately. Survival times are used to generate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment.  
     [0272] 3.2. Established Disease Model  
     [0273] Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student&#39;s t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value≦0.05 compared to the vehicle control group.  
     [0274] 3.3. Orthotopic Disease Models  
     [0275] 3.3.1. Mammary Fat Pad Assay  
     [0276] Tumor cells or fragments, of mammary adenocarcinoma origin, are implanted directly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student&#39;s t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group.  
     [0277] Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value≦0.05 compared to the vehicle control group. In addition, this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student&#39;s t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment.  
     [0278] 3.3.2. Intraprostatic Assay  
     [0279] Tumor cells or fragments, of prostatic adenocarcinoma origin, are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents. The prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles. The successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student&#39;s t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student&#39;s t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment.  
     [0280] 3.3.3. Intrabronchial Assay  
     [0281] Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea. The trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student&#39;s t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student&#39;s t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment.  
     [0282] 3.3.4. Intracecal Assay  
     [0283] Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizing the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student&#39;s t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student&#39;s t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment.  
     [0284] 4. Secondary (Metastatic) Antitumor Efficacy  
     [0285] 4.1. Spontaneous Metastasis  
     [0286] Tumor cells are inoculated s.c. and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student&#39;s t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment for both of these endpoints.  
     [0287] 4.2. Forced Metastasis  
     [0288] Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student&#39;s t-test after conducting an F-test, with significance at p≦0.05 compared to the vehicle control group in the experiment for both endpoints.  
    
     
       
         1 
         
           
             4  
           
           
             1  
             498  
             DNA  
             Homo sapiens  
           
            1 

atggtcaacc ccaccgtgtt cttccacatc tctgtcgacg gtgagtcctt gggccgcatc     60 

tcttttgagc tgtttgcaga caagtttcca aagacagcag aaaacttttg tgctctgaat    120 

actggagaga aaggatttgg ttacaagggt tgctgctttc acagaattat tccagggttt    180 

atgtgtcatg gtggtgactt cacacaccat aatggcactg gtggcaagtc aatctacggg    240 

gagaaagttg atgatgacaa cttcatcctg aagcatacag gtcctggcat attgtccatg    300 

gcaaatgctg gacccaacac aaatggttcc cagtttttca tctgcactgc caagtctgag    360 

tggttggata gcaagcatgt ggtcattggc aaggtgaaag aaggcatgaa tattgtggag    420 

gccatggagc actttgggtc caggaatggc aagaccagca agaaggtcac cattcctgac    480 

tttggacaac tcgaataa                                                  498 

 
           
             2  
             164  
             PRT  
             Homo sapiens  
           
            2 

Met Val Asn Pro Thr Val Phe Phe His Ile Ser Val Asp Gly Glu Ser 
1               5                   10                  15 

Leu Gly Arg Ile Ser Phe Glu Leu Phe Ala Asp Lys Phe Pro Lys Thr 
            20                  25                  30 

Ala Glu Asn Phe Cys Ala Leu Asn Thr Gly Glu Lys Gly Phe Gly Tyr 
        35                  40                  45 

Lys Gly Cys Cys Phe His Arg Ile Ile Pro Gly Phe Met Cys His Gly 
    50                  55                  60 

Gly Asp Phe Thr His His Asn Gly Thr Gly Gly Lys Ser Ile Tyr Gly 
65                  70                  75                  80 

Glu Lys Val Asp Asp Asp Asn Phe Ile Leu Lys His Thr Gly Pro Gly 
                85                  90                  95 

Ile Leu Ser Met Ala Asn Ala Gly Pro Asn Thr Asn Gly Ser Gln Phe 
            100                 105                 110 

Phe Ile Cys Thr Ala Lys Ser Glu Trp Leu Asp Ser Lys His Val Val 
        115                 120                 125 

Ile Gly Lys Val Lys Glu Gly Met Asn Ile Val Glu Ala Met Glu His 
    130                 135                 140 

Phe Gly Ser Arg Asn Gly Lys Thr Ser Lys Lys Val Thr Ile Pro Asp 
145                 150                 155                 160 

Phe Gly Gln Leu 

 
           
             3  
             164  
             PRT  
             Homo sapiens  
           
            3 

Val Asn Pro Thr Val Phe Phe Asp Ile Ala Val Asp Gly Glu Pro Leu 
1               5                   10                  15 

Gly Arg Val Ser Phe Glu Leu Phe Ala Asp Lys Val Pro Lys Thr Ala 
            20                  25                  30 

Glu Asn Phe Arg Ala Leu Ser Thr Gly Glu Lys Gly Phe Gly Tyr Lys 
        35                  40                  45 

Gly Ser Cys Phe His Arg Ile Ile Pro Gly Phe Met Cys Gln Gly Gly 
    50                  55                  60 

Asp Phe Thr Arg His Asn Gly Thr Gly Gly Lys Ser Ile Tyr Gly Glu 
65                  70                  75                  80 

Lys Phe Glu Asp Glu Asn Phe Ile Leu Lys His Thr Gly Pro Gly Ile 
                85                  90                  95 

Leu Ser Met Ala Asn Ala Gly Pro Asn Thr Asn Gly Ser Gln Phe Phe 
            100                 105                 110 

Ile Cys Thr Ala Lys Thr Glu Trp Leu Asp Gly Lys His Val Val Phe 
        115                 120                 125 

Gly Lys Val Lys Glu Gly Met Asn Ile Val Glu Ala Met Glu Arg Phe 
    130                 135                 140 

Gly Ser Arg Asn Gly Lys Thr Ser Lys Lys Ile Thr Ile Ala Asp Cys 
145                 150                 155                 160 

Gly Gln Leu Glu 

 
           
             4  
             308  
             PRT  
             Homo sapiens  
           
            4 

Met Gly His Leu Thr Pro Val Ala Ala Pro Arg Leu Ala Cys Ala Phe 
1               5                   10                  15 

Val Pro Thr Asn Ala Gln Arg Arg Ala Thr Ala Lys Arg Lys Leu Glu 
            20                  25                  30 

Arg Gln Leu Glu Arg Arg Ala Lys Gln Ala Lys Arg Arg Arg Ile Leu 
        35                  40                  45 

Thr Ile Val Gly Gly Ser Leu Ala Ala Val Ala Val Ile Val Ala Val 
    50                  55                  60 

Val Val Thr Val Val Val Asn Lys Asp Asp His Gln Ser Thr Thr Ser 
65                  70                  75                  80 

Ala Thr Pro Thr Asp Ser Ala Ser Thr Ser Pro Pro Gln Ala Ala Thr 
                85                  90                  95 

Ala Pro Pro Leu Pro Pro Phe Lys Pro Ser Ala Asn Leu Gly Ala Asn 
            100                 105                 110 

Cys Gln Tyr Pro Pro Ser Pro Asp Lys Ala Val Lys Pro Val Lys Leu 
        115                 120                 125 

Pro Arg Thr Gly Lys Val Pro Thr Asp Pro Ala Gln Val Ser Val Ser 
    130                 135                 140 

Met Val Thr Asn Gln Gly Asn Ile Gly Leu Met Leu Ala Asn Asn Glu 
145                 150                 155                 160 

Ser Pro Cys Thr Val Asn Ser Phe Val Ser Leu Ala Gln Gln Gly Phe 
                165                 170                 175 

Phe Lys Gly Thr Thr Cys His Arg Leu Thr Thr Ser Pro Met Leu Ala 
            180                 185                 190 

Val Leu Gln Cys Gly Asp Pro Lys Gly Asp Gly Thr Gly Gly Pro Gly 
        195                 200                 205 

Tyr Gln Phe Ala Asn Glu Tyr Pro Thr Asp Gln Tyr Ser Ala Asn Asp 
    210                 215                 220 

Pro Lys Leu Asn Glu Pro Val Ile Tyr Pro Arg Gly Thr Leu Ala Met 
225                 230                 235                 240 

Ala Asn Ala Gly Pro Asn Thr Asn Ser Ser Gln Phe Phe Met Val Tyr 
                245                 250                 255 

Arg Asp Ser Lys Leu Pro Pro Gln Tyr Thr Val Phe Gly Thr Ile Gln 
            260                 265                 270 

Ala Asp Gly Leu Thr Thr Leu Asp Lys Ile Ala Lys Ala Gly Val Ala 
        275                 280                 285 

Gly Gly Gly Glu Asp Gly Lys Pro Ala Thr Glu Val Thr Ile Thr Ser 
    290                 295                 300 

Val Leu Leu Asp 
305