EP1268804A2 - Proteine helicoidale zalpha51 - Google Patents
Proteine helicoidale zalpha51Info
- Publication number
- EP1268804A2 EP1268804A2 EP01916704A EP01916704A EP1268804A2 EP 1268804 A2 EP1268804 A2 EP 1268804A2 EP 01916704 A EP01916704 A EP 01916704A EP 01916704 A EP01916704 A EP 01916704A EP 1268804 A2 EP1268804 A2 EP 1268804A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- amino acid
- residues
- acid residues
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 108090000623 proteins and genes Proteins 0.000 title claims description 187
- 102000004169 proteins and genes Human genes 0.000 title claims description 129
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 240
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 233
- 229920001184 polypeptide Polymers 0.000 claims abstract description 230
- 238000000034 method Methods 0.000 claims abstract description 110
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 100
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 78
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 78
- 239000002157 polynucleotide Substances 0.000 claims abstract description 78
- 230000004927 fusion Effects 0.000 claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims description 232
- 210000001519 tissue Anatomy 0.000 claims description 78
- 239000012472 biological sample Substances 0.000 claims description 46
- 239000002773 nucleotide Substances 0.000 claims description 43
- 125000003729 nucleotide group Chemical group 0.000 claims description 43
- 102000004127 Cytokines Human genes 0.000 claims description 36
- 108090000695 Cytokines Proteins 0.000 claims description 35
- 230000000295 complement effect Effects 0.000 claims description 29
- 210000004185 liver Anatomy 0.000 claims description 27
- 239000000523 sample Substances 0.000 claims description 26
- 230000002068 genetic effect Effects 0.000 claims description 25
- 230000027455 binding Effects 0.000 claims description 22
- 239000007795 chemical reaction product Substances 0.000 claims description 22
- 239000013604 expression vector Substances 0.000 claims description 22
- 238000012360 testing method Methods 0.000 claims description 22
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 19
- 238000013518 transcription Methods 0.000 claims description 18
- 230000035897 transcription Effects 0.000 claims description 18
- 150000001413 amino acids Chemical class 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 17
- 210000005228 liver tissue Anatomy 0.000 claims description 16
- 241000124008 Mammalia Species 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- 230000005856 abnormality Effects 0.000 claims description 9
- 230000004807 localization Effects 0.000 claims description 8
- 239000013610 patient sample Substances 0.000 claims description 8
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 7
- 210000004748 cultured cell Anatomy 0.000 claims description 7
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 6
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 238000009396 hybridization Methods 0.000 claims description 6
- 208000018360 neuromuscular disease Diseases 0.000 claims description 6
- 230000033001 locomotion Effects 0.000 claims description 5
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims description 4
- 239000002853 nucleic acid probe Substances 0.000 claims description 4
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 claims description 3
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 abstract description 12
- 238000011160 research Methods 0.000 abstract description 8
- 239000000463 material Substances 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 120
- 108020004414 DNA Proteins 0.000 description 80
- 108020004635 Complementary DNA Proteins 0.000 description 50
- 239000013598 vector Substances 0.000 description 41
- 238000003556 assay Methods 0.000 description 39
- 238000010804 cDNA synthesis Methods 0.000 description 39
- 239000002299 complementary DNA Substances 0.000 description 39
- 206010028980 Neoplasm Diseases 0.000 description 38
- 230000014509 gene expression Effects 0.000 description 38
- 241000282414 Homo sapiens Species 0.000 description 33
- 230000000694 effects Effects 0.000 description 32
- 239000000203 mixture Substances 0.000 description 30
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 28
- 125000003275 alpha amino acid group Chemical group 0.000 description 28
- 235000001014 amino acid Nutrition 0.000 description 27
- 241000700605 Viruses Species 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 23
- 239000002609 medium Substances 0.000 description 23
- 102000005962 receptors Human genes 0.000 description 23
- 108020003175 receptors Proteins 0.000 description 23
- 238000006467 substitution reaction Methods 0.000 description 20
- 239000012634 fragment Substances 0.000 description 19
- 239000013612 plasmid Substances 0.000 description 19
- 238000004458 analytical method Methods 0.000 description 18
- 241000701161 unidentified adenovirus Species 0.000 description 18
- 241001465754 Metazoa Species 0.000 description 17
- 238000001727 in vivo Methods 0.000 description 17
- 239000013615 primer Substances 0.000 description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 16
- 230000003248 secreting effect Effects 0.000 description 15
- 230000009261 transgenic effect Effects 0.000 description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000005557 antagonist Substances 0.000 description 14
- 239000012091 fetal bovine serum Substances 0.000 description 14
- 238000000338 in vitro Methods 0.000 description 14
- 239000003112 inhibitor Substances 0.000 description 14
- 238000001890 transfection Methods 0.000 description 14
- 238000011282 treatment Methods 0.000 description 14
- 108020004705 Codon Proteins 0.000 description 13
- 230000000875 corresponding effect Effects 0.000 description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 12
- 206010027476 Metastases Diseases 0.000 description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 12
- 201000011510 cancer Diseases 0.000 description 12
- 201000010099 disease Diseases 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 11
- 108010000521 Human Growth Hormone Proteins 0.000 description 11
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 11
- 239000003814 drug Substances 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 11
- 239000006166 lysate Substances 0.000 description 11
- 230000009401 metastasis Effects 0.000 description 11
- 102000002265 Human Growth Hormone Human genes 0.000 description 10
- 239000000854 Human Growth Hormone Substances 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 239000000427 antigen Substances 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 239000003550 marker Substances 0.000 description 10
- 230000001717 pathogenic effect Effects 0.000 description 10
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 10
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 9
- 210000004556 brain Anatomy 0.000 description 9
- 230000008859 change Effects 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 9
- 230000002759 chromosomal effect Effects 0.000 description 9
- 238000003776 cleavage reaction Methods 0.000 description 9
- 229940079593 drug Drugs 0.000 description 9
- 235000013601 eggs Nutrition 0.000 description 9
- 108020001507 fusion proteins Proteins 0.000 description 9
- 102000037865 fusion proteins Human genes 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 150000002632 lipids Chemical class 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 244000052769 pathogen Species 0.000 description 9
- 230000007017 scission Effects 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 238000011830 transgenic mouse model Methods 0.000 description 9
- 230000014616 translation Effects 0.000 description 9
- 241000699660 Mus musculus Species 0.000 description 8
- 230000004071 biological effect Effects 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- 210000002950 fibroblast Anatomy 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- 238000003780 insertion Methods 0.000 description 8
- 230000037431 insertion Effects 0.000 description 8
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 8
- 210000004072 lung Anatomy 0.000 description 8
- 210000004962 mammalian cell Anatomy 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000003612 virological effect Effects 0.000 description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 108090000157 Metallothionein Proteins 0.000 description 7
- 206010029260 Neuroblastoma Diseases 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 230000004913 activation Effects 0.000 description 7
- 230000003321 amplification Effects 0.000 description 7
- 230000033115 angiogenesis Effects 0.000 description 7
- 230000036755 cellular response Effects 0.000 description 7
- 238000003745 diagnosis Methods 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 239000003102 growth factor Substances 0.000 description 7
- 230000003394 haemopoietic effect Effects 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 239000002502 liposome Substances 0.000 description 7
- 229920002521 macromolecule Polymers 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 230000008685 targeting Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 108091029865 Exogenous DNA Proteins 0.000 description 6
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 6
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 6
- 102000003792 Metallothionein Human genes 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- 239000000556 agonist Substances 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 235000011089 carbon dioxide Nutrition 0.000 description 6
- 230000012292 cell migration Effects 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 6
- 230000002950 deficient Effects 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 210000003527 eukaryotic cell Anatomy 0.000 description 6
- 238000013508 migration Methods 0.000 description 6
- 238000003908 quality control method Methods 0.000 description 6
- 230000010076 replication Effects 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 6
- 229910001868 water Inorganic materials 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 5
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000024245 cell differentiation Effects 0.000 description 5
- 210000000349 chromosome Anatomy 0.000 description 5
- 231100000433 cytotoxic Toxicity 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 230000002500 effect on skin Effects 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 210000002889 endothelial cell Anatomy 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 239000005090 green fluorescent protein Substances 0.000 description 5
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 238000013507 mapping Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 229940054269 sodium pyruvate Drugs 0.000 description 5
- 238000001356 surgical procedure Methods 0.000 description 5
- 239000003053 toxin Substances 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 238000013519 translation Methods 0.000 description 5
- 241000701447 unidentified baculovirus Species 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 102000003951 Erythropoietin Human genes 0.000 description 4
- 108090000394 Erythropoietin Proteins 0.000 description 4
- 101150112014 Gapdh gene Proteins 0.000 description 4
- 229920002683 Glycosaminoglycan Polymers 0.000 description 4
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 4
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 102000004877 Insulin Human genes 0.000 description 4
- 108090001061 Insulin Proteins 0.000 description 4
- 102000004889 Interleukin-6 Human genes 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 4
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 108091061960 Naked DNA Proteins 0.000 description 4
- 206010061309 Neoplasm progression Diseases 0.000 description 4
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 102000004243 Tubulin Human genes 0.000 description 4
- 108090000704 Tubulin Proteins 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 230000003171 anti-complementary effect Effects 0.000 description 4
- 230000000692 anti-sense effect Effects 0.000 description 4
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 4
- 210000001638 cerebellum Anatomy 0.000 description 4
- 230000002490 cerebral effect Effects 0.000 description 4
- 210000001612 chondrocyte Anatomy 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 230000000120 cytopathologic effect Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000003511 endothelial effect Effects 0.000 description 4
- 230000006539 extracellular acidification Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 229940125396 insulin Drugs 0.000 description 4
- 229940100601 interleukin-6 Drugs 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 4
- 229960000485 methotrexate Drugs 0.000 description 4
- 238000000520 microinjection Methods 0.000 description 4
- 230000005012 migration Effects 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 230000008488 polyadenylation Effects 0.000 description 4
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 4
- 238000000159 protein binding assay Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 210000000278 spinal cord Anatomy 0.000 description 4
- 230000004936 stimulating effect Effects 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000001131 transforming effect Effects 0.000 description 4
- 238000012384 transportation and delivery Methods 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 230000005751 tumor progression Effects 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- 108020005345 3' Untranslated Regions Proteins 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241001260012 Bursa Species 0.000 description 3
- 102000000844 Cell Surface Receptors Human genes 0.000 description 3
- 108010001857 Cell Surface Receptors Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 241000702421 Dependoparvovirus Species 0.000 description 3
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 3
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 3
- 102000006992 Interferon-alpha Human genes 0.000 description 3
- 108010047761 Interferon-alpha Proteins 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 102000000588 Interleukin-2 Human genes 0.000 description 3
- -1 JJL-2 Proteins 0.000 description 3
- 229930182816 L-glutamine Natural products 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 241001452677 Ogataea methanolica Species 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 208000032107 Rigor Mortis Diseases 0.000 description 3
- 108010084592 Saporins Proteins 0.000 description 3
- 241000256251 Spodoptera frugiperda Species 0.000 description 3
- 102000036693 Thrombopoietin Human genes 0.000 description 3
- 108010041111 Thrombopoietin Proteins 0.000 description 3
- 102000004338 Transferrin Human genes 0.000 description 3
- 108090000901 Transferrin Proteins 0.000 description 3
- 102400001320 Transforming growth factor alpha Human genes 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 239000003708 ampul Substances 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000007385 chemical modification Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 238000002405 diagnostic procedure Methods 0.000 description 3
- 229940105423 erythropoietin Drugs 0.000 description 3
- 239000003797 essential amino acid Substances 0.000 description 3
- 235000020776 essential amino acid Nutrition 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000009545 invasion Effects 0.000 description 3
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 210000003101 oviduct Anatomy 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 210000001322 periplasm Anatomy 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 239000002987 primer (paints) Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000002027 skeletal muscle Anatomy 0.000 description 3
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 210000001550 testis Anatomy 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- 239000012581 transferrin Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000005748 tumor development Effects 0.000 description 3
- 230000002792 vascular Effects 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- PDRJLZDUOULRHE-ZETCQYMHSA-N (2s)-2-amino-3-pyridin-2-ylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=N1 PDRJLZDUOULRHE-ZETCQYMHSA-N 0.000 description 2
- DFZVZEMNPGABKO-ZETCQYMHSA-N (2s)-2-amino-3-pyridin-3-ylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CN=C1 DFZVZEMNPGABKO-ZETCQYMHSA-N 0.000 description 2
- FQFVANSXYKWQOT-ZETCQYMHSA-N (2s)-2-azaniumyl-3-pyridin-4-ylpropanoate Chemical compound OC(=O)[C@@H](N)CC1=CC=NC=C1 FQFVANSXYKWQOT-ZETCQYMHSA-N 0.000 description 2
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- XWHHYOYVRVGJJY-QMMMGPOBSA-N 4-fluoro-L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(F)C=C1 XWHHYOYVRVGJJY-QMMMGPOBSA-N 0.000 description 2
- 108020005029 5' Flanking Region Proteins 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 102000014461 Ataxins Human genes 0.000 description 2
- 108010078286 Ataxins Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000201370 Autographa californica nucleopolyhedrovirus Species 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 208000008882 Benign Neonatal Epilepsy Diseases 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 208000014644 Brain disease Diseases 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 206010008025 Cerebellar ataxia Diseases 0.000 description 2
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 2
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 2
- 102000005853 Clathrin Human genes 0.000 description 2
- 108010019874 Clathrin Proteins 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 101710112752 Cytotoxin Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 208000032274 Encephalopathy Diseases 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 2
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 2
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 108010003272 Hyaluronate lyase Proteins 0.000 description 2
- 102000001974 Hyaluronidases Human genes 0.000 description 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 208000006552 Lewis Lung Carcinoma Diseases 0.000 description 2
- 208000009564 MELAS Syndrome Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 108020005196 Mitochondrial DNA Proteins 0.000 description 2
- 201000002169 Mitochondrial myopathy Diseases 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 206010029113 Neovascularisation Diseases 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 108020004485 Nonsense Codon Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 102100038358 Prostate-specific antigen Human genes 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 108020001027 Ribosomal DNA Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 108700025832 Serum Response Element Proteins 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 208000009415 Spinocerebellar Ataxias Diseases 0.000 description 2
- 208000006011 Stroke Diseases 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 206010047631 Vitamin E deficiency Diseases 0.000 description 2
- 238000002679 ablation Methods 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000001261 affinity purification Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 201000004562 autosomal dominant cerebellar ataxia Diseases 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 201000008181 benign familial infantile epilepsy Diseases 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000007975 buffered saline Substances 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 125000000837 carbohydrate group Chemical group 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000011748 cell maturation Effects 0.000 description 2
- 230000009087 cell motility Effects 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- 230000004640 cellular pathway Effects 0.000 description 2
- 230000014107 chromosome localization Effects 0.000 description 2
- 238000002983 circular dichroism Methods 0.000 description 2
- 229930193282 clathrin Natural products 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 108010057085 cytokine receptors Proteins 0.000 description 2
- 231100000599 cytotoxic agent Toxicity 0.000 description 2
- 239000002619 cytotoxin Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000000447 dimerizing effect Effects 0.000 description 2
- 230000006334 disulfide bridging Effects 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 208000005053 encephalomalacia Diseases 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 210000003238 esophagus Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013861 fat-free Nutrition 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 102000013361 fetuin Human genes 0.000 description 2
- 108060002885 fetuin Proteins 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 230000009395 genetic defect Effects 0.000 description 2
- 208000016361 genetic disease Diseases 0.000 description 2
- 238000012254 genetic linkage analysis Methods 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 229960002773 hyaluronidase Drugs 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 125000001165 hydrophobic group Chemical group 0.000 description 2
- 229960002591 hydroxyproline Drugs 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 238000005462 in vivo assay Methods 0.000 description 2
- 208000023692 inborn mitochondrial myopathy Diseases 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 208000006443 lactic acidosis Diseases 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 208000012268 mitochondrial disease Diseases 0.000 description 2
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 2
- 230000001338 necrotic effect Effects 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000037434 nonsense mutation Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 229920002704 polyhistidine Polymers 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000004952 protein activity Effects 0.000 description 2
- 230000012846 protein folding Effects 0.000 description 2
- 210000000449 purkinje cell Anatomy 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 210000003705 ribosome Anatomy 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 210000003497 sciatic nerve Anatomy 0.000 description 2
- 208000018655 severe necrosis Diseases 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 210000002437 synoviocyte Anatomy 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 210000003437 trachea Anatomy 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- UJXJZOCXEZPHIE-YFKPBYRVSA-N (2s)-2-(2-hydroxyethylamino)-4-sulfanylbutanoic acid Chemical compound OCCN[C@H](C(O)=O)CCS UJXJZOCXEZPHIE-YFKPBYRVSA-N 0.000 description 1
- FXGZFWDCXQRZKI-VKHMYHEASA-N (2s)-5-amino-2-nitramido-5-oxopentanoic acid Chemical compound NC(=O)CC[C@@H](C(O)=O)N[N+]([O-])=O FXGZFWDCXQRZKI-VKHMYHEASA-N 0.000 description 1
- QVQOGNOOAMQKCE-ZTYVOHGWSA-N (2s)-n-[(2s)-1-[(2-amino-2-oxoethyl)amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]-1-[(10r,13s,16s,19s,22s)-13-(2-amino-2-oxoethyl)-16-(3-amino-3-oxopropyl)-19-benzyl-22-[(4-methoxyphenyl)methyl]-12,15,18,21,24-pentaoxo-7,8-dithia-11,14,17,20,23-pen Chemical compound C1=CC(OC)=CC=C1C[C@H]1C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)CSSC2(CCCCC2)CC(=O)N1 QVQOGNOOAMQKCE-ZTYVOHGWSA-N 0.000 description 1
- CCAIIPMIAFGKSI-DMTCNVIQSA-N (2s,3r)-3-hydroxy-2-(methylazaniumyl)butanoate Chemical compound CN[C@@H]([C@@H](C)O)C(O)=O CCAIIPMIAFGKSI-DMTCNVIQSA-N 0.000 description 1
- NMWKYTGJWUAZPZ-WWHBDHEGSA-N (4S)-4-[[(4R,7S,10S,16S,19S,25S,28S,31R)-31-[[(2S)-2-[[(1R,6R,9S,12S,18S,21S,24S,27S,30S,33S,36S,39S,42R,47R,53S,56S,59S,62S,65S,68S,71S,76S,79S,85S)-47-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-18-(4-aminobutyl)-27,68-bis(3-amino-3-oxopropyl)-36,71,76-tribenzyl-39-(3-carbamimidamidopropyl)-24-(2-carboxyethyl)-21,56-bis(carboxymethyl)-65,85-bis[(1R)-1-hydroxyethyl]-59-(hydroxymethyl)-62,79-bis(1H-imidazol-4-ylmethyl)-9-methyl-33-(2-methylpropyl)-8,11,17,20,23,26,29,32,35,38,41,48,54,57,60,63,66,69,72,74,77,80,83,86-tetracosaoxo-30-propan-2-yl-3,4,44,45-tetrathia-7,10,16,19,22,25,28,31,34,37,40,49,55,58,61,64,67,70,73,75,78,81,84,87-tetracosazatetracyclo[40.31.14.012,16.049,53]heptaoctacontane-6-carbonyl]amino]-3-methylbutanoyl]amino]-7-(3-carbamimidamidopropyl)-25-(hydroxymethyl)-19-[(4-hydroxyphenyl)methyl]-28-(1H-imidazol-4-ylmethyl)-10-methyl-6,9,12,15,18,21,24,27,30-nonaoxo-16-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29-nonazacyclodotriacontane-4-carbonyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-3-carboxy-1-[[(2S)-1-[[(2S)-1-[[(1S)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@H](Cc4ccccc4)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CSSC[C@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](Cc4ccccc4)NC3=O)[C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc3ccccc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N3CCC[C@H]3C(=O)N[C@@H](C)C(=O)N2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)C(C)C)C(=O)N[C@@H](Cc2c[nH]cn2)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C(=O)N[C@@H](C)C(O)=O NMWKYTGJWUAZPZ-WWHBDHEGSA-N 0.000 description 1
- 108700005160 1-(1-mercaptocyclohexaneacetic acid)-2-(O- methyl-L-tyrosine)-8-L-arginine- vasopressin Proteins 0.000 description 1
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 description 1
- WUAPFZMCVAUBPE-NJFSPNSNSA-N 188Re Chemical compound [188Re] WUAPFZMCVAUBPE-NJFSPNSNSA-N 0.000 description 1
- OSBLTNPMIGYQGY-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;boric acid Chemical compound OB(O)O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O OSBLTNPMIGYQGY-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 108020005065 3' Flanking Region Proteins 0.000 description 1
- XEVFXAFXZZYFSX-UHFFFAOYSA-N 3-azabicyclo[2.1.1]hexane-4-carboxylic acid Chemical compound C1C2CC1(C(=O)O)NC2 XEVFXAFXZZYFSX-UHFFFAOYSA-N 0.000 description 1
- JYCQQPHGFMYQCF-UHFFFAOYSA-N 4-tert-Octylphenol monoethoxylate Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCO)C=C1 JYCQQPHGFMYQCF-UHFFFAOYSA-N 0.000 description 1
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 1
- 108010066676 Abrin Proteins 0.000 description 1
- 241000228431 Acremonium chrysogenum Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 102100022987 Angiogenin Human genes 0.000 description 1
- 206010059313 Anogenital warts Diseases 0.000 description 1
- 108010005853 Anti-Mullerian Hormone Proteins 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 1
- 241000222128 Candida maltosa Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 1
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 1
- 206010008805 Chromosomal abnormalities Diseases 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- 208000006154 Chronic hepatitis C Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 206010069729 Collateral circulation Diseases 0.000 description 1
- 208000000907 Condylomata Acuminata Diseases 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 208000014094 Dystonic disease Diseases 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 102100031939 Erythropoietin Human genes 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 208000001382 Experimental Melanoma Diseases 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 102100037362 Fibronectin Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000690301 Homo sapiens Aldo-keto reductase family 1 member C4 Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 101000741885 Homo sapiens Protection of telomeres protein 1 Proteins 0.000 description 1
- 101001116548 Homo sapiens Protein CBFA2T1 Proteins 0.000 description 1
- 101000823955 Homo sapiens Serine palmitoyltransferase 1 Proteins 0.000 description 1
- YZJSUQQZGCHHNQ-UHFFFAOYSA-N Homoglutamine Chemical compound OC(=O)C(N)CCCC(N)=O YZJSUQQZGCHHNQ-UHFFFAOYSA-N 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 241000209027 Ilex aquifolium Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 244000285963 Kluyveromyces fragilis Species 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- SNDPXSYFESPGGJ-UHFFFAOYSA-N L-norVal-OH Natural products CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 1
- HXEACLLIILLPRG-YFKPBYRVSA-N L-pipecolic acid Chemical compound [O-]C(=O)[C@@H]1CCCC[NH2+]1 HXEACLLIILLPRG-YFKPBYRVSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 108010092277 Leptin Proteins 0.000 description 1
- 102000016267 Leptin Human genes 0.000 description 1
- 108700005090 Lethal Genes Proteins 0.000 description 1
- 108010031801 Lipopolysaccharide Receptors Proteins 0.000 description 1
- 102000005482 Lipopolysaccharide Receptors Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 206010068836 Metabolic myopathy Diseases 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 208000034819 Mobility Limitation Diseases 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 101000836210 Mus musculus Somatotropin Proteins 0.000 description 1
- 101100369221 Mus musculus Tfrc gene Proteins 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
- 208000021642 Muscular disease Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 201000009623 Myopathy Diseases 0.000 description 1
- 206010061533 Myotonia Diseases 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 206010072359 Neuromyotonia Diseases 0.000 description 1
- 241000221960 Neurospora Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091008606 PDGF receptors Proteins 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 241001504519 Papio ursinus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000015731 Peptide Hormones Human genes 0.000 description 1
- 108010038988 Peptide Hormones Proteins 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 244000203593 Piper nigrum Species 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 231100000742 Plant toxin Toxicity 0.000 description 1
- 102000011653 Platelet-Derived Growth Factor Receptors Human genes 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108010050808 Procollagen Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100038745 Protection of telomeres protein 1 Human genes 0.000 description 1
- 101000762949 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) Exotoxin A Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000007660 Residual Neoplasm Diseases 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 102100022068 Serine palmitoyltransferase 1 Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 206010072148 Stiff-Person syndrome Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 206010042573 Superovulation Diseases 0.000 description 1
- 239000008049 TAE buffer Substances 0.000 description 1
- 239000008051 TBE buffer Substances 0.000 description 1
- 102100036407 Thioredoxin Human genes 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102100034195 Thrombopoietin Human genes 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 102000006747 Transforming Growth Factor alpha Human genes 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 241000255993 Trichoplusia ni Species 0.000 description 1
- 208000037280 Trisomy Diseases 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 244000301083 Ustilago maydis Species 0.000 description 1
- 235000015919 Ustilago maydis Nutrition 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- VWQVUPCCIRVNHF-OUBTZVSYSA-N Yttrium-90 Chemical compound [90Y] VWQVUPCCIRVNHF-OUBTZVSYSA-N 0.000 description 1
- JRMSLDWZFJZLAS-UHFFFAOYSA-M [7-(dimethylamino)-1,9-dimethylphenothiazin-3-ylidene]-dimethylazanium;chloride Chemical compound [Cl-].CC1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC(C)=C3N=C21 JRMSLDWZFJZLAS-UHFFFAOYSA-M 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 230000000689 aminoacylating effect Effects 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 108010072788 angiogenin Proteins 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000037037 animal physiology Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000002391 anti-complement effect Effects 0.000 description 1
- 239000000868 anti-mullerian hormone Substances 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 108010008730 anticomplement Proteins 0.000 description 1
- 229940034982 antineoplastic agent Drugs 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 201000006491 bone marrow cancer Diseases 0.000 description 1
- 239000005388 borosilicate glass Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 230000006041 cell recruitment Effects 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 230000010001 cellular homeostasis Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 108091008394 cellulose binding proteins Proteins 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 230000035572 chemosensitivity Effects 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000001142 circular dichroism spectrum Methods 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 239000000512 collagen gel Substances 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000027326 copulation Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000009223 counseling Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 230000009547 development abnormality Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 208000010118 dystonia Diseases 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000002346 endotoxic effect Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 201000009277 hairy cell leukemia Diseases 0.000 description 1
- 210000000259 harderian gland Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 210000003566 hemangioblast Anatomy 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 210000004024 hepatic stellate cell Anatomy 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 102000054751 human RUNX1T1 Human genes 0.000 description 1
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- MWFRVMDVLYIXJF-BYPYZUCNSA-N hydroxyethylcysteine Chemical compound OC(=O)[C@@H](N)CSCCO MWFRVMDVLYIXJF-BYPYZUCNSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 210000001865 kupffer cell Anatomy 0.000 description 1
- HXEACLLIILLPRG-RXMQYKEDSA-N l-pipecolic acid Natural products OC(=O)[C@H]1CCCCN1 HXEACLLIILLPRG-RXMQYKEDSA-N 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 210000002414 leg Anatomy 0.000 description 1
- 229940039781 leptin Drugs 0.000 description 1
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 210000000415 mammalian chromosome Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- CWWARWOPSKGELM-SARDKLJWSA-N methyl (2s)-2-[[(2s)-2-[[2-[[(2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s)-5-amino-2-[[(2s)-1-[(2s)-6-amino-2-[[(2s)-1-[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-5 Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)OC)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CCCN=C(N)N)C1=CC=CC=C1 CWWARWOPSKGELM-SARDKLJWSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 208000024191 minimally invasive lung adenocarcinoma Diseases 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 230000002297 mitogenic effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000014399 negative regulation of angiogenesis Effects 0.000 description 1
- 230000035407 negative regulation of cell proliferation Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000003961 neuronal insult Effects 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000016087 ovulation Effects 0.000 description 1
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 238000012510 peptide mapping method Methods 0.000 description 1
- 210000003668 pericyte Anatomy 0.000 description 1
- 208000028169 periodontal disease Diseases 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000003123 plant toxin Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 210000000064 prostate epithelial cell Anatomy 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000003156 radioimmunoprecipitation Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000001525 receptor binding assay Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 230000026267 regulation of growth Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 208000037803 restenosis Diseases 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 108091008601 sVEGFR Proteins 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000010845 search algorithm Methods 0.000 description 1
- 238000002805 secondary assay Methods 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 229960004509 serum gonadotrophin Drugs 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 108010018381 streptavidin-binding peptide Proteins 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- NPDBDJFLKKQMCM-UHFFFAOYSA-N tert-butylglycine Chemical compound CC(C)(C)C(N)C(O)=O NPDBDJFLKKQMCM-UHFFFAOYSA-N 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000012250 transgenic expression Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 230000009452 underexpressoin Effects 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 210000001177 vas deferen Anatomy 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 230000000982 vasogenic effect Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000007998 vessel formation Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003357 wound healing promoting agent Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
Definitions
- Cytokines are polypeptide hormones that are produced by a cell and affect cell growth or metabolism in either autocrine, paracrine or endocrine fashion. In multicellular animals, cytokines control cell growth, migration, differentiation, and maturation. Cytokines play a role in both normal development and pathogenesis, including the development of solid tumors.
- Cytokines are physicochemically diverse, ranging in size from 5 kDa (TGF- ⁇ ) to 140 kDa (Mullerian-inhibiting substance). Structurally, cytokines include a group distinguished by their four-helix bundle conformation. They include single polypeptide chains, as well as disulfide-linked homodimers and heterodimers.
- Cytokines influence cellular events by binding to cell-surface receptors. Binding initiates a chain of signalling events within the cell, which ultimately resalts in phenotypic changes such as cell division, protease production, cell migration, expression of cell surface proteins, and production of additional growth factors.
- cytokines erythropoietin, thrombopoietin, and G-CSF stimulate the production of erythrocytes, platelets, and neutrophils, respectively, from precursor cells in the bone marrow.
- Development of mature cells from pluripotent progenitors may require the presence of a plurality of factors.
- Interferon-alpha is used in the treatment of hairy cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, condylomata acuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal and
- Cytokines and Cytokine Receptors An Overview
- PDGF Platelet-derived growth factor
- the hematopoietic cytokine erythropoietin has been developed for the treatment of anemias (e.g., EP 613,683).
- G- CSF, GM-CSF, IFN- ⁇ , IFN- ⁇ , and IL-2 have also been approved for use in humans (Aggarwal and Puri, ibid.).
- Experimental evidence supports additional therapeutic uses of cytokines and their inhibitors. Inhibition of PDGF receptor activity has been shown to reduce intimal ⁇ yperplasia in injured baboon arteries (Giese et al., Restenosis Summit NHL Poster Session #23, 1996; U.S. Patent No. 5,620,687).
- VEGFs Vascular endothelial growth factors
- a soluble VEGF receptor (soluble flt-1) has been found to block binding of VEGF to cell-surface receptors and to inhibit the growth of vascular tissue in vitro (Biotechnology News 16(17):5-6, 1996).
- Experimental evidence suggests that inhibition of angiogenesis may be used to block tumor development (Biotechnology News, Nov. 13, 1997) and that angiogenesis is an early indicator of cervical cancer (Br. J. Cancer 76:1410-1415, 1997).
- thrombopoietin has been shown to stimulate the production of platelets in vivo (Kaushansky et al., Nature 369:568-571, 1994) and has been the subject of several clinical trials (reviewed by von dem Borne et al., Bailliere's Clin. Haematol. 11:427-445, 1998).
- Cytokines are used in the laboratory to study developmental processes, and in laboratory and industry settings as components of cell culture media.
- the figure is a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2.
- the profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters.
- affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
- Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzvmol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu- Glu affinity tag (Grussenmeyer et al., Proc. Natl.
- allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
- allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
- arnino-terminal ⁇ and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
- a "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCAC 3' is complementary to 5' GTGCAT 3*.
- corresponding to when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned.
- degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and
- GAC triplets each encode Asp).
- expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
- isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
- isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
- Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985)
- an "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
- the isolated polypeptide or protein is substantially free of other polypeptides or proteins, particularly those of animal origin. It is preferred to provide the polypeptides and proteins in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
- the term “isolated” does not exclude the presence of the same polypeptide or protein in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
- “Operably linked” means that two or more entities are joined together such that they function in concert for their intended purposes.
- DNA segments the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator.
- “operably linked” includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired function(s) of the sequences are retained.
- ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
- a "polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
- bp base pairs
- nt nucleotides
- kb kilobases
- the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
- polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
- promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
- a “protein” is a macromolecule comprising one or more polypeptide chains.
- a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless, thus, a protein "consisting of, for example, from 15 to 1500 amino acid residues may further contain one or more carbohydrate chains.
- a “secretory signal sequence” is a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
- the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
- a “segment” is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes.
- a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.
- the present invention provides novel cytokine polypeptides and proteins.
- This novel cytokine termed "zalpha51" was identified by the presence of polypeptide and polynucleotide features characteristic of four-helix bundle cytokines
- erythropoietin e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin, and growth hormone.
- SEQ ID NO:2 Analysis of the amino acid sequence shown in SEQ ID NO:2 indicates the presence of four amphipathic, alpha-helical regions. These regions include at least amino acid residues 43 through 57 (helix A), 98 through 112 (helix B), 126 through 140 (helix C), and 192 through 206 (helix D). Within these helical regions, residues that are expected to lie within the core of the four-helix bundle occur at positions 43, 46, 47, 50, 53, 54,
- Inter-helix loops comprise approximately residues 58 through 97 (loop A/B), residues 113 through 125 (loop B/C) and 141 through 191 (loop C D) as shown on SEQ ID NO: 2, with corresponding nucleotide sequence shown in SEQ ID NO: 1.
- the human zalpha51 cDNA (SEQ ID NO:l) encodes a polypeptide of at least 232 amino acid residues. This sequence is predicted to include a secretory peptide of at least 14 residues.
- Polypeptides of the present invention comprise at least 6, preferably at least 9, more preferably at least 15 contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO: 5.
- the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, up to the entire predicted mature polypeptide (residues 18 to 196 of SEQ ID NO:2) or the primary translation product (residues 1 to 198 of SEQ ID NO:2).
- the primary translation product will include residues 1- 243 as shown in SEQ ID NO: 5.
- these polypeptides can further comprise additional, non-zalpha51, polypeptide sequence(s).
- the helical regions of the zalpha51 polypeptides must include from 15 to 23 contiguous amino acid residues comprising residues 54 (Ala) to 60 (Glu) as shown in SEQ ID NO: 5 (helix A); from 15 to 26 contiguous amino acid residues comprising residues 109 (He) to 114 (Gin) as shown in SEQ ID NO: 5 (helix B); from 15 to 23 contiguous amino acid residues comprising residues 146 (Asp) to 151 (Leu) as shown in SEQ ID NO: 5 (helix C); and from 15 to 27 contiguous amino acid residues comprising residues 205 (Arg) to 217 (Ala) as shown in SEQ ID NO: 5 (helix D).
- helical regions of zalpha51 can include amino acid residues 38 (Leu) to 60 (Glu) as shown in SEQ ID NO: 5 (helix A); amino acid residues 91 (Ser) to 114 (Gin) as shown in SEQ ID NO: 5 (helix B); amino acid residues 136 (Gin) to 158 (Ala) as shown in SEQ T-D NO: 5 (helix C); and amino acid residues 203 (Thr) to 227 (Ala) as shown in SEQ ID NO: 5 (helix D).
- the corresponding nucleotides encoding these regions can be found in SEQ ID NOS: 4 and 6.
- polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:2.
- An "epitope” is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984.
- Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least nine amino acid residues in length.
- Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666, 1983.
- Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells or tissue samples.
- Antibodies to linear epitopes are also useful for detecting fragments of zalpha51, such as might occur in body fluids or cell culture media.
- Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zalpha51 protein. It is preferred that the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided, and are described herein.
- polypeptides that comprise the entire four-helix bundle of a zalpha51 polypeptide (e.g., residues 43-170 of SEQ ID NO:2) or portions thereof, including amino acid residues 43 through 57 (helix A), 98 through 112 (helix B), 126 through 140 (helix C), and 156 through 170 (helix D).
- polypeptides may further comprise all or part of one or both of the native zalpha51 amino-terminal (residues 18-42 of SEQ ID NO:2); carboxyl-terminal (residues 171-232 of SEQ J-D NO:2) regions, as well as non-zalpha51 amino acid residues or polypeptide sequences as disclosed in more detail below.
- the present invention are polypeptides that comprise the residues 38-227 of SEQ ID NO:5, or portions thereof, including amino acid residues 38 through 60 (helix A), 91 through 114 (helix B), 136 through 158 (helix C), and 203 through 227 (helix D).
- Such polypeptides may further comprise all or part of one or both of the native zalpha51 amino-terminal (residues 29-37 of SEQ ID NO: 5); carboxyl-terminal (residues 228-243 of SEQ J-D NO:5) region.
- Polypeptides of the present invention can be prepared with one or more amino acid substitutions, deletions or additions as compared to SEQ ID NO:2. These changes are preferably of a minor nature, that is conservative amino acid substitutions and other changes that do not significantly affect the folding or activity of the protein or polypeptide, and include amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, an amino or carboxyl-terminal cysteine residue to facilitate subsequent linking to maleimide-activated keyhole limpet hemocyanin, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as disclosed above. Two or more affinity tags may be used in combination.
- Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zalpha51 polypeptide and the affinity tag.
- Preferred cleavage sites include thrombin cleavage sites and factor Xa cleavage sites.
- the helical domains of zalpha51 will be useful for preparing chimeric fusion molecules, particularly with other four-helix bundle cytokines to determine and modulate receptor binding specificity.
- fusion proteins engineered with helix A and/or helix D and fusion proteins that combine helical and loop domains from other four-helix bundle cytokines such as JJL-2, erythropoietin, and G-CSF.
- loops can comprise approximately residues 58 through 97 (loop A/B), residues 113 through 125 (loop B/C), and 141 through 191 (loop C/D) from SEQ ID. NO: 2.
- variants will require loop domains comprising residues 61-90 (loop A/B), residues 115-135 (loop B/C), and residues 159- 202 (loop C/D), all shown in SEQ ID NO: 5.
- the present invention further provides a variety of other polypeptide fusions.
- a zalpha51 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
- Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
- Immunoglobulin-zalpha51 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zalpha51 analogs.
- a zalpha51 polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a multi-functional molecule.
- One or more helices of a zalpha51 polypeptide can be joined to another cytokine to enhance or otherwise modify its biological properties.
- Auxiliary domains can be fused to zalpha51 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
- a zalpha51 polypeptide or protein can be targeted to a predetermined cell type by fusing a zalpha51 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
- a zalpha51 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
- Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al.. Connective Tissue Research 34:1-9, 1996.
- Polypeptide fusions of the present invention will generally contain not more than about 1,500 amino acid residues, preferably not more than about 1,200 residues, more preferably not more than about 1,000 residues, and will in many cases be considerably smaller.
- a zalpha51 polypeptide of 215 residues can be fused to E. coli -galactosidase (1,021 residues; see Casadaban et al, J. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xa cleavage site.
- residues 18-232 of SEQ ID NO:2 can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.
- the polypeptides of the present invention comprise at least nine contiguous residues of SEQ ID NO:2. These polypeptides may further comprise additional residues as shown in SEQ J-D NO:2, a variant of SEQ J-D NO:2, or another protein as disclosed herein.
- the resulting polypeptide certain embodiments will be at least 90%, other embodiments will be at least 95%, 96%, 97%, 98%, or 99% identical to the corresponding region of SEQ ID NO: 2. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919. 1992.
- the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math.
- FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
- the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
- the present invention includes polypeptides having one or more conservative amino acid changes as compared with the amino acid sequence of SEQ ID NO:2.
- the BLOSUM62 matrix (Table 1) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, ibid.). Thus, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention.
- the term "conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
- Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least one 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
- the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
- Non-naturally occurring amino acids include, without limitation, tran ⁇ .-3-methylproline, 2,4-methanoproline, -.-4-hydroxyproline, tr ⁇ /M-4- hydroxyproline, N-methylglycine, /Zo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
- Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins.
- an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor fR ⁇ As.
- Methods for synthesizing amino acids and aminoacylating tR ⁇ A are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. c li S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; ⁇ llman et al., Methods ⁇ nzvmol. 202:301, 1991; Chung et al., Science 259:806-_809, 1993; and Chung et al.,
- coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
- the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.
- Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
- Amino acid sequence changes are made in zalpha51 polypeptides so as to minimize disruption of higher order structure essential to biological activity.
- Amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can identify specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
- Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity, secondary structure propensities, binary patterns, complementary packing, and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996).
- determination of structure will be accompanied by evaluation of activity of modified molecules.
- changes in amino acid residues will be made so as not to disrupt the four- helix bundle structure of the protein family.
- the effects of amino acid sequence changes can be predicted by, for example, computer modeling using available software (e.g., the Insight JJ® viewer and homology modeling tools; MSI, San Diego, CA) or determined by analysis of crystal structure (see, e.g., Lapthorn et al, Nature 369:455- 461, 1994; Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995).
- Protein folding can be measured by circular dichroism (CD).
- a hydrophilicity profile of SEQ JJD NO:2 is shown in the attached figure. Those skilled in the art will recognize that this hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a zalpha51 polypeptide, so as not to disrupt the overall profile. Residues within the core of the four-helix bundle can be replaced with a hydrophobic residue selected from the group consisting of Leu, JJe, Val, Met, Phe, Tip, Gly. The residues predicted to be on the exposed surface of the four-helix bundle will be relatively intolerant of substitution.
- the length and amino acid composition of the interdomain loops are also expected to be important for receptor binding (and therefore biological activity); conservative substitutions and relatively small insertions and deletions are thus preferred within the loops, and the insertion of bulky amino acid residues (e.g., Phe) will in general be avoided.
- Essential amino acids in the polypeptides of the present invention can be identified experimentally according to procedures known in the art, such as site- directed mutagenesis or alanine-scan ing mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al.. Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991).
- site- directed mutagenesis or alanine-scan ing mutagenesis Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al.. Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991.
- single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule.
- variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations.
- This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes. In many cases, the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a zalpha51 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide.
- expressing the complete zalpha51 sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved.
- Differential processing of individual chains may result in heterogeneity of expressed polypeptides.
- Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zalpha51 variant polypeptides.
- Assays that can be scaled up for high throughput include mitogenesis assays, which can be run in a 96-well format.
- Mutagenized DNA molecules that encode active zalpha51 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
- polypeptide fragments or variants of SEQ ID NO:2 that retain the activity of wild-type zalpha51.
- the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zalpha51 polypeptides disclosed above.
- a representative DNA sequence encoding the amino acid sequence of SEQ JJD NO:2 is shown in SEQ ID NO:l.
- SEQ ID NO:3 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha51 polypeptide of SEQ J-D NO: 2.
- the degenerate sequence of SEQ J-D NO:3 also provides all RNA sequences encoding SEQ ID NO:2 by substituting U for T.
- zalpha51 polypeptide-encoding polynucleotides comprising nucleotides 1 or 52 nucleotides 696 of SEQ ID NO:3, and their RNA equivalents are contemplated by the present invention, as are segments of SEQ ID NO: 3 encoding other zalpha51 polypeptides disclosed herein.
- Table 2 sets forth the one-letter codes used within SEQ J-D NO:3 to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter. “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C. TABLE 2
- degenerate codons used in SEQ ID NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 3, below.
- Gap One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
- the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
- WSN can, in some circumstances, encode arginine
- MGN degenerate codon for arginine
- AGY serine
- some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be readily tested for functionality as described herein.
- the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l or a sequence complementary thereto under stringent conditions.
- stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
- T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
- Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
- the isolated polynucleotides of the present invention include DNA and RNA.
- Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zalpha51 RNA. Liver cells are preferred. Fibroblasts are another preferred source.
- Total RNA can be prepared using guanidine HC1 extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972).
- cDNA Complementary DNA
- poly(A) + RNA using known methods. J-n the alternative, genomic DNA can be isolated. Polynucleotides encoding zalpha51 polypeptides are then identified and isolated by, for example, hybridization or PCR.
- Full-length clones encoding zalpha51 can be obtained by conventional cloning procedures.
- Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
- Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
- Expression libraries can be probed with antibodies to zalpha51, receptor fragments, or other specific binding partners.
- Zalpha51 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zalpha51 gene.
- Promoter elements from a zalpha51 gene can thus be used to direct the expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zalpha51 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670.
- an endogenous zalpha51 gene in a cell is altered by introducing into the zalpha51 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
- the targeting sequence is a zalpha51 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zalpha51 locus, whereby the sequences within the construct become operably linked with the endogenous zalpha51 coding sequence.
- an endogenous zalpha51 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
- sequences disclosed in SEQ ID NOS:l and 2 represent a single allele of human zalpha51. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
- the present invention further provides counterpart polypeptides and polynucleotides from other species ("orthologs").
- zalpha51 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
- Orthologs of human zalpha51 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
- a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zalpha51 as disclosed above.
- a library is then prepared from mRNA of a positive tissue or cell line.
- a zalpha51 -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence.
- a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zalpha51 sequence disclosed herein.
- the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zalpha51 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
- any zalpha51 polypeptide including variants and fusion proteins
- one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 3 and 4, above.
- those of skill in the art can use standard software to devise zalpha51 variants based upon the nucleotide and amino acid sequences described herein.
- the present invention thus provides ⁇ a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO:l, SEQ ID NO:2, and portions thereof.
- Suitable forms of computer-readable media include magnetic media and optically-readable media.
- magnétique media examples include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIPTM disk.
- Optically readable media are exemplified " by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD- recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
- compact discs e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD- recordable
- DVD digital versatile/video discs
- the zalpha51 polypeptides of the present invention can be produced according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide.
- cells into which have been introduced an expression vector include both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA.
- Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells.
- a DNA sequence encoding a zalpha51 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
- the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
- a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
- the secretory signal sequence may be that of zalpha51, or may be derived from another secreted protein (e.g., t-PA; see, U.S. Patent No. 5,641,655) or synthesized de novo.
- the secretory signal sequence is operably linked to the zalpha51 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
- Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
- Cultured mammalian cells can be used as hosts within the present invention.
- Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb,
- Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61; or CHO DG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines.
- COS-1 ATCC No. CRL 1650
- COS-7 ATCC No. CRL 1651
- BHK ATCC No. CRL 1632
- BHK 570 ATCC No. CRL 10314
- 293 ATCC No. CRL 1573
- Graham et al. J. Gen. Virol. 36:59-72, 1977
- Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. J-n general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
- Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
- Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited with the American Type Culture Collection, Manassas, VA USA under accession numbers 98669 and 98668, respectively, and derivatives thereof.
- Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants.”
- a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
- Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
- a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
- Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
- hygromycin resistance multi-drug resistance
- puromycin acetyltransferase can also be used.
- the adenovirus system can also be used for protein production in vitro.
- the cells By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
- adenovirus vector-infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cvtotechnol. 15:145-55, 1994).
- an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell.
- non-secreted proteins can also be effectively obtained.
- Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) according to methods known in the art.
- recombinant baculovirus is produced through the use of a transposon-based system described by Luckow et al. (J. Virol 67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (Bac-to-BacTM kit; Life Technologies, Rockville, MD).
- the transfer vector (e.g., pFasfBaclTM; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551- 1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.
- transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above. Using techniques known in the art, a transfer vector containing a zalpha51 -encoding sequence is transformed into
- E. coli host cells and the cells are screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
- the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, such as Sf9 cells.
- Recombinant virus that expresses zalpha51 protein is subsequently produced.
- Recombinant viral stocks are made by methods commonly used the art.
- the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveTM cells; J-nvitrogen, Carlsbad, CA). See, for example, U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers.
- the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
- MOI multiplicity of infection
- Procedures used are generally known in the art.
- Other higher eukaryotic cells can also be used as hosts, including plant cells and avian cells.
- Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11 :47- 58, 1987.
- Fungal cells, including yeast cells can also be used within the present invention.
- Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
- Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
- Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
- a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
- Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
- Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a zalpha51 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
- the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
- the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
- the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
- Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
- suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. 4
- the growth medium will generally select for cells ⁇ containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
- Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
- polypeptides and proteins of the present invention it is preferred to purify the polypeptides and proteins of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
- a purified polypeptide or protein is substantially free of other polypeptides or proteins, particularly those of animal origin.
- Expressed recombinant zalpha51 proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques.
- Proteins comprising a polyhistidine affinity tag are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid. Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
- Zalpha51 polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, J-RL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.
- zalpha51 proteins can be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non- pegylated; and may or may not include an initial methionine amino acid residue.
- Target cells for use in zalpha51 activity assays include, without limitation, vascular cells (especially endothelial cells and smooth muscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts (including human dermal fibroblasts and lung fibroblasts), fetal lung cells, articular synoviocytes, pericytes, chondrocytes, osteoblasts, and prostate epithelial cells.
- vascular cells especially endothelial cells and smooth muscle cells
- hematopoietic (myeloid and lymphoid) cells include, without limitation, liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts (including human dermal fibroblasts and lung fibroblasts), fetal lung cells, articular synoviocytes, pericytes,
- Zalpha51 proteins of the present invention are characterized by their activity, that is, modulation of the proliferation, differentiation, migration, adhesion, or metabolism of responsive cell types.
- Biological activity of zalpha51 proteins is assayed using in vitro or in vivo assays designed to detect cell proliferation, differentiation, migration or adhesion; or changes in cellular metabolism (e.g., production of other growth factors or other macromolecules).
- Many suitable assays are known in the art, and representative assays are disclosed herein.
- Assays using cultured cells are most convenient for screening, such as for determining the effects of amino acid substitutions, deletions, or insertions.
- in vivo assays will generally be employed to confirm and further characterize biological activity.
- Certain in vitro models such as the three-dimensional collagen gel matrix model of Pepper et al. (Biochem. Biophys. Res. Comm. 189:824-831, 1992), are sufficiently complex to assay histological effects.
- Assays can be performed using exogenously produced proteins, or may be carried out in vivo or in vitro using cells expressing the polypeptide(s) of interest.
- Assays can be conducted using zalpha51 proteins alone or in combination with other growth factors, such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative assays are disclosed below.
- growth factors such as members of the VEGF family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representative assays are disclosed below.
- Activity of zalpha51 proteins can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to an appropriate animal model.
- Assays measuring cell proliferation or differentiation are well known in the art.
- assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (as disclosed by, e.g., Raines and Ross, Methods Enzymol. 109:749-773, 1985; Wahl et al, Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al., Analytical Biochem.
- Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
- Zalpha51 activity may also be detected using assays designed to measure zalpha51 -induced production of one or more additional growth factors or other macromolecules.
- Preferred such assays include those for determining the presence of hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF ⁇ ), interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF), angiogenin, and other macromolecules produced by the liver.
- Suitable assays include mitogenesis assays using target cells responsive to the macromolecule of interest, receptor-binding assays, competition binding assays, immunological assays (e.g., ELISA), and other formats known in the art.
- Metalloprotease secretion is measured from treated primary human dermal fibroblasts, synoviocytes and chondrocytes.
- the relative levels of collagenase, gelatinase and stromalysin produced in response to culturing in the presence of a zalpha51 protein is measured using zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1_:96- 106, 1990).
- Procollagen collagen synthesis by dermal fibroblasts and chondrocytes in response to a test protein is measured using 3 H-proline incorporation into nascent secreted collagen.
- 3 H-labeled collagen is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J. Biol.
- GAG Glycosaminoglycan secretion from dermal fibroblasts and chondrocytes is measured using a 1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim. Biophvs. Acta 883:173-177, 1986). Collagen and GAG assays are also carried out in the presence of JJ -lo. or TGF- ⁇ to examine the ability of zalpha51 protein to modify the established responses to these cytokines.
- Monocyte activation assays are carried out (1) to look for the ability of zalpha51 proteins to further stimulate monocyte activation, and (2) to examine the ability of zalpha51 proteins to modulate attachment-induced or endotoxin-induced monocyte activation (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987).
- DL-l ⁇ and TNF levels produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA).
- Monocyte/macrophage cells by virtue of CD14 (LPS receptor), areakily sensitive to endotoxin, and proteins with moderate levels of endotoxin-like activity will activate these cells.
- Hematopoietic activity of zalpha51 proteins can be assayed on various hematopoietic cells in culture.
- Preferred assays include primary bone marrow colony assays and later stage lineage-restricted colony assays, which are known in the art (e.g., Holly et al., WJPO Publication WO 95/21920).
- Marrow cells plated on a suitable semi-solid medium e.g., 50% methylcellulose containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotic mix
- Mitogenic activity of zalpha51 polypeptides on hematopoietic cell lines can be measured as disclosed above. Cell migration is assayed essentially as disclosed by Kahler et al.
- a protein is considered to be chemotactic if it induces migration of cells from an area of low protein concentration to an area of high protein concentration.
- a typical assay is performed using modified Boyden chambers with a polystryrene membrane separating the two chambers (Transwell; Corning Costar Corp.). The test sample, diluted in medium containing 1% BSA, is added to the lower chamber of a 24-well plate containing Transwells. Cells are then placed on the Transwell insert that has been pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37°C.
- Non-migrating cells are wiped off the top of the Transwell membrane, and cells attached to the lower face of the membrane are fixed and stained with 0.1% crystal violet. Stained cells are then extracted with 10% acetic acid and absorbance is measured at 600 nm. Migration is then calculated from a standard calibration curve. Cell migration can also be measured using the matrigel method of Grant et al. ("Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial-Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997).
- Cell adhesion activity is assayed essentially as disclosed by LaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are coated with the test protein, non-specific sites are blocked with BSA, and cells (such as smooth muscle cells, leukocytes, or endothelial cells) are plated at a density of approximately 10 4 - 10 5 cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then non-adherent cells are removed by gentle washing. Adhered cells are quantitated by conventional methods (e.g. ⁇ by staining with crystal violet, lysing the cells, and determining the optical density of the lysate).
- Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin.
- a known adhesive protein such as fibronectin or vitronectin.
- the activity of zalpha51 proteins can be measured with a silicon-based biosensor microphysiometer that measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
- An exemplary such device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
- a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257:1906-1912, 1992; Pitchford et al., Meth. Enzvmol.
- the microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zalpha51 proteins, their agonists, and antagonists.
- the microphysiometer is used to measure responses of a zalpha51 -responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zalpha51 polypeptide.
- Zalpha51- responsive eukaryotic cells comprise cells into which a receptor for zalpha51 has been transfected, thereby creating a cell that is responsive to zalpha51, as well as cells naturally responsive to zalpha51.
- Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zalpha51 polypeptide, relative to a control not exposed to zalpha51 are a direct measurement of zalpha51 -modulated cellular responses.
- the present invention thus provides methods of identifying agonists and antagonists of zalpha51 proteins, comprising providing cells responsive to a zalpha51 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells.
- the change in cellular response is shown as a measurable change in extracellular acidification rate.
- Culturing a third portion of the cells in the presence of a zalpha51 protein and the absence of a test compound provides a positive control for the zalpha51 -responsive cells and a control to compare the agonist activity of a test compound with that of the zalpha51 polypeptide.
- Antagonists of zalpha51 can be identified by; exposing the cells to zalpha51 protein in the presence and absence of the test compound, whereby a reduction in zalpha51- stimulated activity is indicative of antagonist activity in the test compound.
- Expression of zalpha51 polynucleotides in animals provides models for further study of the biological effects of overproduction or inhibition of protein activity in vivo.
- Zalpha51 -encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.
- viral delivery systems include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
- Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53,
- Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co- transfected plasmid.
- the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (e.g., the human 293 cell line).
- the host cell e.g., the human 293 cell line.
- adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
- An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963- 967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
- Transgenic mice engineered to express a zalpha51 gene, and mice that exhibit a complete absence of zalpha51 gene function, referred to as "knockout mice” (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-742, 1993). These mice can be employed to study the zalpha51 gene and the protein encoded thereby in an in vivo system. Transgenic mice are particularly useful for investigating the role of zalpha51 proteins in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or underexpression of a specific factor.
- Transgenic mice expressing zalpha51 had severe locomotion disabilities. Microscopic examination of the brain found severe necrosis in the cerebellar folia (encephalomalacia). The necrotic cells were primarily located in the granular and Purkinje cell layers of the cerebellum. Acute perivasculitis was observed in the pons and cerebellar folia of the mice and in the ventral spinal cord of one of the mice. No significant changes were found in the tissues of the nontransgenic control.
- the lesions in the zalpha51 transgenic mice were similar to lesions observed in chicks with vitamin E deficiency (Riddell, Avian Histopathology, p.76. AAVP, Kennett Square, PA, 1987.)
- a form of spinocerebellar ataxia in humans has been associated with a severe deficiency of this vitamin.
- the rapid onset of rigor mortis in these transgenic mice suggests a metabolic derangement.
- Mitochondrial disorders can cause metabolism-related changes in a variety of tissue (e.g. the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke)).
- zalpha51 may have some role in inducing apoptosis, based on the cell death observed in the cerebellum and other tissues. See, Heffner and Schochet, "Skeletal muscle” in Anderson's Pathology, tenth edition, Damjanov and Linder, eds., pp 2653-2690. Mosby Year-Book, Inc., St. Louis, 1990 and Cotran, Kumar, and Robbins, Pathologic Basis of Disease, fifth edition, pp 418-419. W.B. Saunders Co., Philadelphia, 1994.
- inflammation which is associated with many of the cytokines, has been identified as a secondary contributor to certain neurodegenerative diseases (Owens et al., Nature Medicine, 7:161-166, 2001.)
- a loss of normal inhibitory control of muscle contraction has been associated with damage or perturbation of selected gamma-aminobutryric acid- secreting neurons.
- Stiff Man Syndrome exhibit remarkable stiffness of musculature, believed to be mediated through interference of the functioning of their garrmia-aminobutryric acid (GAB A) producing neurons.
- GAB A garrmia-aminobutryric acid
- Other related neuromuscular disorders include myotonia, metabolic myopathies, Isaac's syndrome, dystonia, and tetanic spasms (Valldeoriola, J. Neurol 246:423-431, 1999). These disorders exhibit phenotypic similarities to those seen in zalpha51 -expressing mice, and antagonists of zalpha51 can serve as candidate
- direct measurement of zalpha51 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression, increases in invasiveness and motility of cells, or the gain or loss of expression of zalpha51 in a pre-cancerous or cancerous condition, in comparison to normal tissue, can serve as a diagnostic for transformation, invasion and metastasis in tumor progression.
- knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient.
- Methods of measuring gain and loss of expression are well known in the art and described herein and can be applied to zalpha51 expression.
- polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999).
- zalpha51 gain or loss of expression may serve as a diagnostic for liver, neuroblastoma, endothelial, brain, and other cancers.
- PSA prostate specific antigen
- increased levels of zalpha51 polypeptides, or anti-zalpha51 antibodies in a patient, relative to a normal control can be indicative of liver, neuroblastoma, endothelial, brain, and other cancers (See, e.g., Mulders, TMT, et al., Eur. J. Surgical Oncol. 16:37-41, 1990).
- zalpha51 expression appears to be restricted to liver, neuroblastoma, endothelial, brain, and other cancers in normal human tissues
- lack of zalpha51 expression in liver or strong zalpha51 expression in non-liver tissue would serve as a diagnostic of an abnormality in the cell or tissue type, of invasion or metastasis of cancerous liver tissue into non-liver tissue, and could aid a physician in directing further testing or investigation, or aid in directing therapy.
- zalpha51 is liver-specific
- polynucleotide probes, anti- zalpha51 antibodies, and detection the presence of zalpha51 polypeptides in tissue can be used to assess whether liver tissue is present, for example, after surgery involving the excision of a diseased or cancerous liver or neuronal tissue.
- the ⁇ polynucleotides, polypeptides, and antibodies of the present invention can be used as an aid to determine whether all liver-derived tissue is excised after surgery, for example, after surgery for liver, neuroblastoma, endothelial, brain, and other cancers. In such instances, it is especially important to remove all potentially diseased tissue to maximize recovery from the cancer, and to minimize recurrence.
- Preferred embodiments include fluorescent, radiolabeled, or calorimetrically labeled anti- zalpha51 antibodies and zalpha51 polypeptide binding partners, that can be used histologically or in situ. Moreover, the activity and effect of zalpha51 on tumor progression and metastasis can be measured in vivo.
- Several syngeneic mouse models have been developed to study the influence of polypeptides, compounds or other treatments on tumor progression. In these models, tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models. Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No.
- CRL- 1642 CRL- 1642
- B16 melanoma ATCC No. CRL-6323
- These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro. Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice.
- the Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328,1994).
- C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one-time injection of recombinant adenovirus.
- an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one-time injection of recombinant adenovirus.
- 10 to 10 cells are implanted under the dorsal skin.
- the cells themselves may be infected with recombinant adenovirus, such as one expressing zalpha51, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically.
- the mice normally develop visible tumors within 5 days. The tumors are allowed to grow for a
- the implanted cells can be transiently transfected with zalpha51.
- Use of stable zalpha51 transfectants as well as use of induceable promoters to activate zalpha51 expression in vivo are known in the art and can be used in this system to assess z*** induction of metastasis.
- purified zalpha51 or zalpha51- conditioned media can be directly injected in to this mouse model, and hence be used in this system.
- O'Reilly MS et al. Cell 79:315-328, 1994
- Rusciano D et al. Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.
- Antisense methodology can be used to inhibit zalpha51 gene transcription to examine the effects of such inhibition in vivo.
- Polynucleotides that are complementary to a segment of a zalpha51 -encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ID NO:l
- Such antisense oligonucleotides can also be used to inhibit expression of zalpha51 polypeptide- encoding genes in cell culture.
- Zalpha51 and inhibitors of zalpha51 activity are expected to have a variety of therapeutic applications. These therapeutic applications include treatment of diseases which require immune regulation, including autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, and diabetes. Zalpha51 may be important in the regulation of inflammation, and therefore would be useful in treating rheumatoid arthritis, asthma and sepsis.
- Zalpha51 may be useful in modulating the immune system, whereby zalpha51 and zalpha51 antagonists may be used for reducing graft rejection, preventing graft- vs-host disease, boosting immunity to infectious diseases, treating immunocompromised patients (e.g., HJN + patients), or in improving vaccines.
- Zalpha51 polypeptides can be administered alone or in combination with other vasculogenic or angiogenic agents, including NEGF.
- the two compounds can be administered simultaneously or sequentially as appropriate for the specific condition being treated.
- zalpha51 proteins are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
- pharmaceutical formulations will include a zalpha51 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, or the like.
- a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water, or the like.
- Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
- Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
- Zalpha51 will preferably be used in a concentration of about 10 to 100 ⁇ g/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 ⁇ g/ml may be used.
- the protein will be applied in the range of 0.1-10 ⁇ g/cm 2 of wound area, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. Dosing is daily or intermittently over the period of treatment. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. Sustained release formulations can also be employed.
- a therapeutically effective amount of zalpha51 is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in hematopoietic or immune function, a significant reduction in morbidity, or a significantly increased histological score.
- Zalpha51 proteins, agonists, and antagonists are useful for modulating the expansion, proliferation, activation, differentiation, migration, or metabolism of responsive cell types, which include both primary cells and cultured cell lines.
- responsive cell types which include both primary cells and cultured cell lines.
- hematopoietic cells mesenchymal cells (including stem cells and mature myeloid and lymphoid cells), endothelial cells, smooth muscle cells, fibroblasts, hepatocytes, neural cells and embryonic stem cells.
- Zalpha51 polypeptides are added to tissue culture media for these cell types at a concentration of about 10 pg/ml to about 100 ng/ml.
- zalpha51 proteins can be advantageously combined with other growth factors in culture media.
- zalpha51 proteins can also be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over- or under-production of zalpha51 protein or in the analysis of cell phenotype.
- Zalpha51 proteins can also be used to identify inhibitors of their activity.
- Test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of zalpha51 protein.
- samples can be tested for inhibition of zalpha51 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zalpha51 -dependent cellular responses.
- zalpha51 -responsive cell lines can be transfected with a reporter gene construct that is responsive to a zalpha51- stimulated cellular pathway.
- Reporter gene constructs of this type are known in the art, and will generally comprise a zalpha51 -activated serum response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase.
- SRE serum response element
- Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zalpha51 on the target cells as evidenced by a decrease in zalpha51 stimulation of reporter gene expression.
- Assays of this type will detect compounds that directly block zalpha51 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding.
- compounds or other samples can be tested for direct blocking of zalpha51 binding to receptor using zalpha51 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like).
- a detectable label e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like.
- Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
- antibodies includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies.
- Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking” them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
- humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
- Antibodies are defined to be specifically binding if they bind to a zalpha51 polypeptide or protein with an affinity at least 10-fold greater than the binding affinity to control (non- zalpha51) polypeptide or protein.
- the affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard,
- polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.
- the immunogenicity of a zalpha51 polypeptide may be increased through the use of an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
- Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zalpha51 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
- the polypeptide immunogen may be a full-length molecule or a portion thereof.
- Jf the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
- a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
- Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to zalpha51 polypeptides, and selection of antibody display libraries in phage or similar vectors (e.g., through the use of immobilized or labeled zalpha51 polypeptide).
- Human antibodies can be produced in transgenic, non- human animals that have been engineered to contain human immunoglobulin genes as disclosed in WD?O Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
- assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zalpha51 polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio- immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
- concurrent immunoelectrophoresis radio-immunoassays, radio- immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
- Antibodies to zalpha51 may be used for affinity purification of the protein, within diagnostic assays for determining circulating levels of the protein; for detecting or quantitating soluble zalpha51 polypeptide as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for irnmunohistochernistry; and as antagonists to block protein activity in vitro and in vivo.
- Antibodies to zalpha51 may also be used for tagging cells that express zalpha51; for affinity purification of zalpha51 polypeptides and proteins; in analytical methods employing FACS; for screening expression libraries; and for generating anti-idiotypic antibodies.
- Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targeting of those compounds to cells expressing receptors for zalpha51.
- Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
- Antibodies of the present invention may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications(e.g., inhibition of cell proliferation). See, in general, Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.
- Polypeptides and proteins of the present invention can be used to identify and isolate receptors.
- Zalpha51 receptors may be involved in growth regulation in the liver, blood vessel formation, and other developmental processes.
- zalpha51 proteins and polypeptides can be immobilized on a column, and membrane preparations run over the column (as generally disclosed in immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202). Proteins and polypeptides can also be radiolabeled (Methods Enzymol., vol. 182, "Guide to Protein Purification", M.
- radiolabeled zalpha51 proteins and polypeptides can be used to clone the cognate receptor in binding assays using cells transfected with an expression cDNA library.
- Zalpha51 gene has been mapped to a chromosomal location of 16pl2.1.
- This chromosomal locus has been identified with several neuromuscular diseases or defects, a phenotype that has been seen with zalpha51 transgenic mice.
- identification of a new gene within that cluster has scientific and medical value by providing a possible candidate gene for an inheritable disease which shows linkage in that chromosomal region. Further elucidation of the role of that chromosomal region facilitates the use of genetics as a diagnostic for neuromuscular disease.
- BFIC benign familial infantile convulsions
- 16pl2-ql2 has been linked to the locus 16pl2-ql2 (Lee et al., Human Genet. 103:608-612, 1998 and Szepetowski et al., Am. J. Hum. Genet. 61:889-898, 1997).
- Brody disease which is characterized by progressive impairment of muscular relaxation, has been localized to chromosome 16 (MacLenna et al., Somat. Cell Molec. Genet. 13:341-346, 1987).
- 16pl2-pl3 has also been identified as the familial neuroblastoma locus, which results in a predisposition for neuroblastoma, the most common form of solid tumors in children.
- Zalpha51 maps to this region of the chromosome, and overexpression in mice resulted in severe neuronal damage in specific regions of the brain, making zalpha51 a candidate gene for involvement in neurological disease, such as neuroblastoma.
- zalpha51 Defects in the zalpha51 gene itself may result in a heritable human disease state.
- Molecules of the present invention such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment of diseases associated with a zalpha51 genetic defect.
- zalpha51 polynucleotide probes are particularly useful for diagnosis of gross chromosomal abnormalities associated with loss of heterogeneity (LOH), chromosome gain (e.g. trisomy), translocation, DNA amplification, and the like.
- LH loss of heterogeneity
- chromosome gain e.g. trisomy
- translocation DNA amplification, and the like.
- zalpha51 polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the zalpha51 chromosomal locus.
- the zalpha51 sequences can be used as diagnostics in forensic DNA profiling. A diagnostic could assist physicians in determining the type of disease and appropriate associated therapy, or assistance in genetic counseling.
- the inventive anti-zalpha51 antibodies, polynucleotides, and polypeptides can be used for the detection of zalpha51 polypeptide, mRNA or anti- zalpha51 antibodies, thus serving as markers and be directly used for detecting or genetic diseases or cancers, as described herein, using methods known in the art and described herein.
- diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient, are known in the art.
- Most diagnostic methods comprise the steps of (i) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (ii) producing a first reaction product by incubating the genetic sample with a zalpha51 polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Nisualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a zalpha51 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (
- a difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre-implantation embryo.
- a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the zalpha51 genetic locus, and the like are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples.
- Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated fromm any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like.
- the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ J-D NO:l, or an RNA equivalent thereof.
- Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
- Mutations associated with the zalpha51 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases
- standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase clea
- Radiation hybrid mapping panels that cover the entire human genome are commercially available, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence- tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest, and the establishment of directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
- STSs sequence- tagged sites
- the precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an ⁇ existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
- Sequence tagged sites can also be used independently for chromosomal localization.
- An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome.
- An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), Ge Bank (National Center for Biological).
- polypeptides, nucleic acid and/or antibodies of the present invention may be used in diagnosis or treatment of disorders associated with cell loss or abnormal cell proliferation (including cancer).
- Labeled zalpha51 polypeptides may be used for imaging tumors or other sites of abnormal cell proliferation.
- Inhibitors of zalpha51 activity include anti- zalpha51 antibodies and soluble zalpha51 receptors, as well as other peptidic and non- peptidic agents (including ribozymes). Such antagonists can be used to block the effects of zalpha51 on cells or tissues. Of particular interest is the use of antagonists of zalpha51 activity in cancer therapy. As early detection methods improve it becomes possible to intervene at earlier times in tumor development, making it feasible to use inhibitors of growth factors to block cell proliferation, angiogenesis, and other events that lead to tumor development and metastasis. Inhibitors are also expected to be useful in adjunct therapy after surgery to prevent the growth of residual cancer cells. Inhibitors can also be used in combination with other cancer therapeutic agents.
- zalpha51 inhibitors include small molecule inhibitors and inactive receptor-binding fragments of zalpha51 polypeptides.
- Inhibitors are formulated for pharmaceutical use as generally disclosed above, taking into account the precise chemical and physical nature of the inhibitor and the condition to be treated. The relevant determinations are within the level of ordinary skill in the formulation art.
- zalpha51 may activate the immune system which would be important in boosting immunity to infectious diseases, treating immunocompromised patients, such as HJN+ patients, or in improving vaccines.
- J-n particular, zalpha51 stimulation or expansion of hematopoietic cells, or their progenitors, would provide therapeutic value in treatment of bacterial or viral infection, and as an anti-neoplastic factor.
- zalpha51 stimulation of the immune response against viral and non- viral pathogenic agents including bacteria, protozoa, and fungi
- the present invention include a method of stimulating an immune response in a mammal exposed to an antigen or pathogen comprising the steps of: (1) determining directly or indirectly the level of antigen or pathogen present in said mammal; (2) administering a composition comprising zalpha51 polypeptide in an acceptable pharmaceutical vehicle; (3) determining directly or indirectly the level of antigen or pathogen in said mammal; and (4) comparing the level of the antigen or pathogen in step 1 to the antigen or pathogen level in step 3, wherein a change in the level is indicative of stimulating an immune response.
- the zalpha51 composition is re-administered.
- the antigen is a B cell tumor; a virus; a parasite or a bacterium.
- the present invention provides a method of stimulating an immune response in a mammal exposed to an antigen or pathogen comprising: (1) determining a level of an antigen- or pathogen-specific antibody; (2) administering a composition comprising zalpha51 polypeptide in an acceptable pharmaceutical vehicle; (3) determining a post administration level of antigen- or pathogen-specific antibody; (4) comparing the level of antibody in step (1) to the level of antibody in step (3), wherein an increase in antibody level is indicative of stimulating an immune response.
- Polynucleotides encoding zalpha51 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zalpha51 activity. If a mammal has a mutated or absent zalpha51 gene, a zalpha51 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zalpha51 polypeptide is introduced in vivo in a viral vector.
- viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
- Defective viruses which entirely or almost entirely lack viral genes, are preferred.
- a defective virus is not infective after introduction into a cell.
- Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
- Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSN1) vector (Kaplitt et al., Molec. Cell. ⁇ eurosci. 2:320-330, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest.
- HSN1 herpes simplex virus 1
- a zalpha51 gene can be introduced in a retroviral vector as described, for example, by Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol.
- the vector can be introduced by liposome-mediated transfection ("lipofection").
- lipofection Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027- 8031, 1988).
- lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages, including molecular targeting of liposomes to specific cells. Directing transfection to particular cell types is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Peptidic and non-peptidic molecules can be coupled to liposomes chemically.
- cells are removed from the body, a vector is introduced into the cells as a naked DNA plasmid, and the transformed cells are re-implanted into the body as disclosed above.
- Antisense methodology can be used to inhibit zalpha51 gene transcription in a patient as generally disclosed above.
- Zalpha51 polypeptides and anti-zalpha51 antibodies can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
- polypeptides or antibodies of the present invention may be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance).
- zalpha51 polypeptides or anti-zalpha51 antibodies, or bioactive fragments or portions thereof can be coupled to ⁇ detectable or cytotoxic molecules and delivered to a mammal having cells, tissues, or organs that express the anti-complementary molecule.
- Suitable detectable molecules can be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like.
- Suitable cytotoxic molecules can be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90.
- polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin.
- cytotoxic drugs such as adriamycin.
- the detectable or cytotoxic molecule may be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
- biotin/streptavidin is an exemplary complementary/anticomplementary pair.
- Polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins may be used for targeted cell or tissue inhibition or ablation, such as in cancer therapy.
- conjugates of a zalpha51 polypeptide and a cytotoxin which can be used to target the cytotoxin to a tumor or other tissue that is undergoing undesired angiogenesis or neovascularization.
- Target cells i.e., those displaying the zalpha51 receptor
- bind the zalpha51 -toxin conjugate which is then internalized, killing the cell.
- the effects of receptor-specific cell killing (target ablation) are revealed by changes in whole animal physiology or through histological examination.
- ligand-dependent, receptor-directed cyotoxicity can be used to enhance understanding of the physiological significance of a protein ligand.
- a preferred such toxin is saporin. Mammalian cells have no receptor for saporin, which is non-toxic when it remains extracellular.
- zalpha51 -cytokine fusion proteins or antibody/fragment-cytokine fusion proteins may be used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers). See, generally, Hornick et al., Blood 89:4437-4447, 1997). In general, cytokines are toxic if administered systemically.
- the described fusion proteins enable targeting of a cytokine to a desired site of action, such as a cell having binding sites for zalpha51, thereby providing an elevated local concentration of cytokine.
- Suitable cytokines for this purpose include, for example, interleukin-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF).
- GM-CSF granulocyte-macrophage colony-stimulating factor
- Such fusion proteins may be used to cause cytokine-induced killing of tumors and other tissues undergoing angiogenesis or neovascularization.
- the present inventions provides, but is not limited to, certain embodiments described herein. I-n one aspect, the present invention provides an isolated polypeptide comprising at least nine contiguous amino acid residues of SEQ ID NO:2. In another embodiment at least nine contiguous amino acid residues of SEQ ID NO: 2 are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein and an immunoglobulin constant region.
- the polypeptides are from 15 to 232 amino acid residues, are at least 30 contiguous residues of SEQ J-D NO:2, comprise residues 43-206 of SEQ ID NO:2, comprise residues 18-232 of SEQ JD NO: 2.
- the present invention provides an isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) residues 1-17 of SEQ ID NO:2; (b) residues 43-57 of SEQ JO NO:2; (c) residues 98-112 of SEQ ID NO:2; (d) residues 126-140 of SEQ ID NO:2; and (e) residues 192- 206 of SEQ ID NO:2.
- the helical regions of the zalpha51 polypeptides must include from 15 to 23 contiguous amino acid residues comprising residues 54 (Ala) to 60 (Glu) as shown in SEQ J-D NO: 5 (helix A); from 15 to 26 contiguous amino acid residues comprising residues 109 (He) to 114 (Gin) as shown in SEQ J-D NO: 5 (helix B); from 15 to 23 contiguous amino acid residues comprising residues 146 (Asp) to 151 (Leu) as shown in SEQ ID NO: 5 (helix C); and from 15 to 27 contiguous amino acid residues comprising residues 205 (Arg) to 217 (Ala) as shown in SEQ ID NO: 5 (helix D).
- helical regions of zalpha51 can include amino acid residues 38 (Leu) to 60 (Glu) as shown in SEQ J-D NO: 5 (helix A); amino acid residues 91 (Ser) to 114 (Gin) as shown in SEQ J-D NO: 5 (helix B); amino acid residues 136 (Gin) to 158 (Ala) as shown in SEQ J-D NO: 5 (helix C); and amino acid residues 203 (Thr) to 227 (Ala) as shown in SEQ J_D NO: 5 (helix D).
- the corresponding nucleotides encoding these regions can be found in SEQ J-D NOS: 4 and 6.
- the present invention includes an isolated polypeptide comprising a sequence of amino acid residues as shown in SEQ J-D NO: 5 from residue 1 to residue 243.
- the present invention includes a fusion polypeptide comprising a four-helix bundle cytokine wherein at least one or more of helices A, B, C, or D within the polypeptide comprise a sequence of amino acid residues selected from the group consisting of: (a) amino acid residues 38 (Leu) to 60 (Glu) as shown in SEQ ID NO: 5; (b) amino acid residues 91 (Ser) to 114 (Gin) as shown in SEQ J-D NO: 5; (c) amino acid residues 136 (Gin) to 158 (Ala) as shown in SEQ J-D NO: 5; and (d) amino acid residues 203 (Thr) to 227 (Ala) as shown in SEQ J-D NO: 5.
- At least two of helices A, B, C, or D within the fusion polypeptide comprises a sequence of amino acids selected from the group consisting of: (a) amino acid residues 38 (Leu) to 60 (Glu) as shown in SEQ ID NO: 5; (b) amino acid residues 91 (Ser) to 114 (Gin) as shown in SEQ J-D NO: 5; (c) amino acid residues 136 (Gin) to 158 (Ala) as shown in SEQ ID NO: 5; and (d) amino acid residues 203 (Thr) to 227
- polynucleotide molecules encoding the polypeptides and fusion polypeptides described herein are provided using the corresponding nucleotide sequences as shown in SEQ J-D NOS: 1, 3, 4, and 6. These nucleotide sequences include polynucleotide molecules as shown in SEQ J-D NO: 4 from nucleotide 35 to nucleotide 766 or SEQ J-D NO: 6 from nucleotide 1 to nucleotide 729.
- the present invention includes expression vectors comprising the following operably linked elements: a transcription promoter; a DNA segments encoding polypeptides described herein, and a transcription terminator.
- cultured cells into which has been introduced the expression vectors, and express the DNA segments are also provided.
- the present invention provides methods of making a protein comprising: culturing a cell into which has been introduced the expression vectors described herein under conditions whereby the DNA segment is expressed and the polypeptide is produced; and recovering the protein from the cell.
- the present invention includes antibodies that specifically binds to the polypeptides described herein.
- the present invention includes methods of using the polynucleotides and polypeptides provided herein. These include a method of detecting the presence of an RNA encoding SEQ J-D NO: 5 in a biological sample, comprising the steps of : (a) contacting a zalpha51 nucleic acid probe under hybridizing conditions with either (i) test RNA molecules from the biological sample, or (ii) nucleic acid molecules synthesized from the RNA molecules, wherein the probe has a nucleotide sequence comprising either a portion of the nucleotide sequence of the nucleic acid molecule of claim 17, or its complement, and (b) detecting the formation of hybrids of the nucleic acid probe with either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of RNA encoding SEQ ID NO: 5 in the biological sample.
- the biological sample is taken from a mammal with a neuromuscular disorder, or the mammal has a loco
- Also included is a method of detecting the presence of a polypeptide as shown in SEQ J-D NO: 5, or portion thereof, in a biological sample comprising the steps of: (a) contacting the biological sample with an antibody, or an antibody fragment, of claim 34, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.
- the present invention includes a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:l or the complement of SEQ J-D NO:l, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the first reaction product; and comparing said first reaction product to a control reaction product from a wild type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.
- Other methods include a method for detecting liver tissue in a patient sample, comprising: obtaining a tissue or biological sample from a patient; incubating the tissue or biological sample with an antibody as described herein under conditions wherein the antibody binds to its complementary polypeptide in the tissue or biological sample; visualizing the antibody bound in the tissue or biological sample; and comparing levels and localization of antibody bound in the tissue or biological sample from the patient to a non-liver control tissue or biological sample, wherein an increase in the level or localization of antibody bound to the patient tissue or biological sample relative to the non-liver control tissue or biological sample is indicative of liver tissue in a patient sample.
- a method for detecting liver tissue in a patient sample comprising: obtaining a tissue or biological sample from a patient; labeling a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:5 or the complement of SEQ ID NO:5; incubating the tissue or biological sample with under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the labeled polynucleotide in the tissue or biological sample; and comparing the level and localization of labeled polynucleotide hybridization in the tissue or biological sample from the patient to a control non-liver tissue or biological sample, wherein an increase in the level or localization of the labeled polynucleotide hybridization to the patient tissue or biological sample relative to the control non-liver tissue or biological sample is indicative of liver tissue in a patient sample.
- An expression plasmid containing all or part of a polynucleotide encoding zalpha51 is constructed via homologous recombination.
- a fragment of zalpha51 cDNA is isolated by PCR using the polynucleotide sequence of SEQ ID NO: 1 with flanking regions at the 5' and 3' ends corresponding to the vector sequences flanking the zalpha51 insertion point.
- the primers for PCR each include from 5' to 3' end: 40 bp of flanking sequence from the vector and 17 bp corresponding to the amino and carboxyl termini from the open reading frame of zalpha51.
- Plamid pZMP6 is a mammalian expression vector containing an expression cassette having the cytomegalovirus immediate early promoter, multiple restriction sites for insertion of coding sequences, a stop codon, and a human growth hormone terminator; an E. coli origin of replication; a mammalian selectable marker expression unit comprising an S V40 promoter, enhancer and origin of replication, a DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae. It was constructed from pZP9 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, under Accession No.
- yeast genetic elements taken from pRS316 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, under Accession No. 77145), an internal ribosome entry site (IRES) element from poliovirus, arid the extracellular domain of CD8 truncated at the C-terminal end of the transmembrane domain.
- IRS internal ribosome entry site
- yeast/DNA mixtures are electropulsed using power supply (BioRad Laboratories, Hercules, CA) settings of 0.75 kV (5 kV/cm), ⁇ ohms, 25 ⁇ F.
- power supply BioRad Laboratories, Hercules, CA
- To each cuvette is added 600 ⁇ l of 1.2 M sorbitol, and the yeast is plated in two 300- ⁇ l aliquots onto two URA-D plates and incubated at 30°C.
- the Ura + yeast transformants from a single plate are resuspended in 1 ml H2O and spun briefly to pellet the yeast cells.
- the cell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
- lysis buffer 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA.
- Five hundred microliters of the lysis mixture is added to an Eppendorf tube containing 300 ⁇ l acid-washed glass beads and 200 ⁇ l phenol-chloroform, vortexed for 1 minute intervals two or three times, and spun for 5 minutes in an Eppendorf centrifuge at maximum speed.
- DH10BTM cells obtained from Life Technologies, Inc., Gaithersburg, MD
- DH10BTM cells obtained from Life Technologies, Inc., Gaithersburg, MD
- the cells are electropulsed at 1.7 kN, 25 ⁇ F, and 400 ohms.
- CHO DG44 cells (Chasin et al, Som. Cell. Molec. Genet. 12:555-666, 1986) are plated in 10-cm tissue culture dishes and allowed to grow to approximately 50% to 70% confluency overnight at 37°C, 5% CO2, in Ham's F12/FBS media (Ham's F12 medium (Life Technologies), 5% fetal bovine serum (Hyclone, Logan, UT), 1% L-glutamine (JRH Biosciences, Lenexa, KS), 1% sodium pyruvate (Life Technologies)).
- the cells are then transfected with the plasmid zalpha51/pZMP6 by liposome-mediated transfection using a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperininecarboxamido)ethyl]-N,N-dimethyl-l- propaniminium-trifluoroacetate and the neutral lipid dioleoyl phosphatidylethanolamine in membrane-filetered water (LipofectamineTM Reagent, Life Technologies), in serum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).
- SF serum free
- Zalpha51/pZMP6 is diluted into 15-ml tubes to a total final volume of 640 ⁇ l with SF media.
- 35 ⁇ l of LipofectamineTM is mixed with 605 ⁇ l of SF medium. The resulting mixture is added to the DNA mixture and allowed to incubate approximately 30 minutes at room temperature.
- Five ml of SF media is added to the DNA:LipofectamineTM mixture.
- the cells are rinsed once with 5 ml of SF media, aspirated, and the DNA:LipofectamineTM mixture is added.
- the cells are incubated at 37°C for five hours, then 6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each plate.
- the plates are incubated at 37°C overnight, and the DNA:LipofectamineTM mixture is replaced with fresh 5% FBS/Ham's media the next day.
- the cells are split into T-175 flasks in growth medium.
- the cells are stained with FITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, CA) followed by anti-FlTC-conjugated magnetic beads (Miltenyi Biotec).
- the CD8-positive cells are separated using commercially available columns (mini-MACS columns; Miltenyi Biotec) according to the manufacturer's directions and put into DMEM/Ham's F12/5% FBS without nucleosides but with 50 nM methotrexate (selection medium).
- Cells are plated for subcloning at a density of 0.5, 1 and 5 cells per well in 96-well dishes in selection medium and allowed to grow out for approximately two weeks. The wells are checked for evaporation of medium and brought back to 200 ⁇ l per well as necessary during this process. When a large percentage of the colonies in the plate are near confluency, 100 ⁇ l of medium is collected from each well for analysis by dot blot, and the cells are fed with fresh selection medium. The supernatant is applied to a nitrocellulose filter in a dot blot apparatus, and the filter is treated at 100°C in a vacuum oven to denature the protein.
- the filter is incubated in 625 mM Tris-glycine, pH 9.1, 5mM ⁇ -mercaptoethanol, at 65°C, 10 minutes, then in 2.5% nonfat dry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4°C on a rotating shaker.
- the filter is incubated with the antibody-HRP conjugate in 2.5% non-fat dry milk Western A buffer for 1 hour at room temperature on a rotating shaker.
- the filter is then washed three times at room temperature in PBS plus 0.01% Tween 20, 15 minutes per wash.
- the filter is developed with chemiluminescence reagents (ECLTM direct labelling kit; Amersham Corp., Arlington Heights, JL) according to the manufacturer's directions and exposed to film (Hyperfilm ECL, Amersham Corp.) for approximately 5 minutes. Positive clones are trypsinized from the 96-well dish and transferred to 6-well dishes in selection medium for scaleup and analysis by Western blot.
- ECLTM direct labelling kit Amersham Corp., Arlington Heights, JL
- film Hyperfilm ECL, Amersham Corp.
- Example 3 Full-length zalpha51 protein is produced in BHK cells transfected with pZMP6/zalpha51 (Example 1).
- BHK 570 cells (ATCC CRL-10314) are plated in 10- cm tissue culture dishes and allowed to grow to approximately 50 to 70% confluence overnight at 37°C, 5% CO 2 , in DMEM/FBS media (DMEM, Gibco/BRL High Glucose; Life Technologies), 5% fetal bovine serum (Hyclone, Logan, UT), 1 mM L- glutamine (JRH Biosciences, Lenexa, KS), 1 mM sodium pyruvate (Life Technologies).
- DMEM Gibco/BRL High Glucose
- 5% fetal bovine serum Hyclone, Logan, UT
- 1 mM L- glutamine JRH Biosciences, Lenexa, KS
- 1 mM sodium pyruvate Life Technologies.
- the cells are then transfected with pZMP6/zalpha51 by liposome- mediated transfection (using LipofectamineTM; Life Technologies), in serum free (SF) media (DMEM supplemented with 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).
- SF serum free
- the plasmid is diluted into 15-ml tubes to a total final volume of 640 ⁇ l with SF media. 35 ⁇ l of the lipid mixture is mixed with 605 ⁇ l of SF medium, and the resulting mixture is allowed to incubate approximately 30 minutes at room temperature. Five milliliters of SF media is then added to the DNA:lipid mixture.
- the cells are rinsed once with 5 ml of SF media, aspirated, and the DNA:lipid mixture is added.
- the cells are incubated at 37°C for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media is added to each plate.
- the plates are incubated at 37°C overnight, and the DNA:lipid mixture is replaced with fresh 5% FBS/DMEM media the next day.
- the cells are split into T-162 flasks in selection medium (DMEM + 5% FBS, 1% L-Gln, 1% NaPyr, 1 ⁇ M methotrexate).
- DMEM + 5% FBS, 1% L-Gln, 1% NaPyr, 1 ⁇ M methotrexate Approximately 10 days post-transfection, two 150-mm culture dishes of methotrexate-resistant colonies from each transfection are trypsinized, and the cells are pooled and plated into a T-162 flask and transferred to large-scale culture
- the protein coding region of human zalpha51 is amplified by PCR using primers that add Pmel and Ascl restriction sites at the 5' and 3' termini respectively. Amplification is performed with a full- length zalpha51 cDNA template in a PCR reaction as follows: one cycle at 95 °C for 5 minutes; followed by 15 cycles at 95°C for 1 min., 61°C for 1 min., and 72°C for 1.5 min.; followed by 72°C for 7 min.; followed by a 4°C soak. The PCR reaction product is loaded onto a 1.2% low-melting-temperature agarose gel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA).
- the zalpha51 PCR product is excised from the gel and purified using a commercially available kit comprising a silica gel mambrane spin column (QIAquick® PCR Purification Kit and gel cleanup kit; Qiagen, Inc.) as per kit instructions.
- the PCR product is then digested with Pmel and Ascl, phenol/chloroform extracted, EtOH precipitated, and rehydrated in 20 ml TE
- Vector pTG12-8 was derived from p2999B4 (Palmiter et al., Mol Cell Biol. 13:5266-5275, 1993) by insertion of a rat insulin II intron (ca. 200 bp) and polylinker (Fse ITPme I Asc I) into the Nru I site.
- the vector comprises a mouse metallothionein (MT-1) promoter (ca.
- cDNA is inserted between the insulin II and hGH sequences.
- Clones containing zalpha51 are identified by plasmid DNA miniprep followed by digestion with Pmel and Ascl. A positive clone is sequenced to insure that there were no deletions or other anomalies in the construct.
- DNA is prepared using a commercially available kit (Maxi Kit, Qiagen, Inc.), and the zalpha51 cDNA is released from the pTG12-8 vector using Pmel and Ascl enzymes.
- the cDNA is isolated on a 1% low melting temperature agarose gel and excised from the gel. The gel slice is melted at 70 ⁇ C, and the DNA is extracted twice with an equal volume of Tris-buffered phenol, precipitated with EtOH, and resuspended in 10 ⁇ l H2O.
- the zalpha51 cDNA is cloned into the EcoRV-AscI sites of a modified pAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998).
- This construct contains the green fluorescent protein (GFP) marker gene.
- the CMV promoter driving GFP expression is replaced with the SV40 promoter, and the SV40 polyadenylation signal is replaced with the human growth hormone polyadenylation signal.
- the native polylinker is replaced with Fsel, EcoRV, and Ascl sites.
- This modified form of pAdTrack-CMV is named pZyTrack. Ligation is performed using a commercially available DNA ligation and screening kit (Fast-Link® kit;
- Clones containing zalpha51 are identified by digestion of mini prep DNA with Fsel and Ascl. In order to linearize the plasmid, approximately 5 ⁇ g of the resulting pZyTrack zalpha51 plasmid is digested with Pmel. Approximately 1 ⁇ g of the linearized plasmid is cotransformed with 200 ng of supercoiled pAdEasy (He et al., ibid.) into E. coli BJ5183 cells (He et al., ibid.). The co-transformation is done using a Bio-Rad Gene Pulser at 2.5 kN, 200 ohms and 25 ⁇ Fa.
- the entire co-transformation mixture is plated on 4 LB plates containing 25 ⁇ g/ml kanamycin.
- the smallest colonies are picked and expanded in LB/kanamycin, and recombinant adenovirus DNA is identified by standard DNA miniprep procedures.
- the recombinant adenovirus miniprep DNA is transformed into E. coli DH10BTM competent cells, and DNA is prepared using a Maxi Kit (Qiagen, Inc.) aaccording to kit instructions.
- Pad enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 ⁇ l containing 20-30U of Pad.
- the digested DNA is extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol.
- the DNA pellet is resuspended in 10 ⁇ l distilled water.
- a T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada), inoculated the day before and grown to 60-70% confluence, is transfected with the Pad digested DNA.
- the Pad-digested DNA is diluted up to a total volume of 50 ⁇ l with sterile HBS (150mM NaCl, 20mM HEPES).
- DOTAP N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethyl-ammonium salts
- the media is removed from the 293A cells and washed with 5 ml serum-free minimum essential medium (MEM) alpha containing ImM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and 25mM HEPES buffer (reagents obtained from Life Technologies, Gaithersburg, MD).
- 5 ml of serum-free MEM is added to the 293A cells and held at 37°C.
- the DNA/lipid mixture is added drop-wise to the T25 flask of 293A cells, mixed gently, and incubated at 37°C for 4 hours. After 4 hours the media containing the DNA/lipid mixture is aspirated off and replaced with 5 ml complete MEM containing 5% fetal bovine serum.
- the transfected cells are monitored for GFP expression and formation of foci (viral plaques).
- the cells Seven days after transfection of 293A cells with the recombinant adenoviral DNA, the cells express the GFP protein and start to form foci (viral "plaques"). The crude viral lysate is collected using a cell scraper to collect all of the
- the lysate is transferred to a 50-ml conical tube.
- the crude lysate is amplified (Primary (1°) amplification) to obtain a working "stock" of zalpha51 rAdV lysate.
- Ten 10cm plates of nearly confluent (80- 90%) 293 A cells are set up 20 hours previously, 200 ml of crude rAdV lysate is added to each 10-cm plate, and the cells are monitored for 48 to 72 hours for CPE (cytopathic effect) under the white light microscope and expression of GFP under the fluorescent microscope.
- CPE cytopathic effect
- a secondary (2°) amplification of zalpha51 rAdV is then performed. Twenty 15-cm tissue culture dishes of 293A cells are prepared so that the cells are 80- 90% confluent. All but 20 ml of 5% MEM media is removed, and each dish is inoculated with 300-500 ml of the 1° amplified rAdv lysate. After 48 hours the 293 A cells are lysed from virus production, the lysate is collected into 250-ml polypropylene centrifuge bottles, and the rAdV is purified. NP-40 detergent is added to a final concentration of 0.5% to the bottles of crude lysate in order to lyse all cells.
- Bottles are placed on a rotating platform for 10 minutes agitating as fast as possible without the bottles falling over.
- the debris is pelleted by centrifugation at 20,000 X G for 15 minutes.
- the supernatant is transferred to 250-ml polycarbonate centrifuge bottles, and 0.5 volume of 20% PEG8000/2.5 M NaCl solution is added.
- the bottles are shaken overnight on ice.
- the bottles are centrifuged at 20,000 X G for 15 minutes, and the supernatant is discarded into a bleach solution.
- Using a sterile cell scraper the white, virus/PEG precipitate from 2 bottles is resuspended in 2.5 ml PBS.
- the resulting virus solution is placed in 2-ml microcentrifuge tubes and centrifuged at 14,000 X G in the microcentrifuge for 10 minutes to remove any additional cell debris.
- the supernatant from the 2-ml microcentrifuge tubes is transferred into a 15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/ml with CsCl.
- the solution is transferred to 3.2-ml, polycarbonate, thick-walled centrifuge tubes and spun at 348,000 X G for 3-4 hours at 25 ⁇ C.
- the virus forms a white band. Using wide-bore pipette tips, the virus band is collected.
- a commercially available ion-exchange columns (e.g., PD-10 columns ⁇ prepacked with Sephadex® G-25M; Pharmacia Biotech, Piscataway, NJ) is used to desalt the virus preparation.
- the column is equilibrated with 20 ml of PBS.
- the virus is loaded and allowed to run into the column.
- 5 ml of PBS is added to the column, and fractions of 8-10 drops are collected.
- the optical densities of 1:50 dilutions of each fraction are determined at 260 nm on a spectrophotometer. Peak fractions are pooled, and the optical density (OD) of a 1:25 dilution is determined.
- glycerol is added to the purified virus to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80 ⁇ C.
- Trangenic animals expressing zalpha51 genes are producing using adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown,
- the donors are acclimated for 1 week and then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma, St.
- hCG (Sigma) IP. to induce superovulation. Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection.
- Copulation is confirmed by the presence of a vaginal plug the morning following mating.
- Fertilized eggs are collected under a surgical scope (Leica MZ12 Stereo
- the oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (Table 4) that has been incubated with 5% CO2, 5% O2, and 90% N2 at 37°C. The eggs are then stored in a
- Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO2 -equilibrated mineral oil.
- the DNA is drawn into an injection needle (pulled from a 0.75mm ID, 1mm OD borosilicate glass capillary), and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei.
- Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected.
- Successfully microinjected eggs are transferred into an organ tissue-culture dish with pregassed W640 medium for storage overnight in a 37°C/5% CO2 incubator.
- the following day 12-17 healthy 2-cell embryos from the previous day's injection are transferred into the recipient.
- the swollen ampulla is located and holding the oviduct between the ampulla and the bursa, a nick in the oviduct is made with a 28 g needle close to the bursa, making sure not to tear the ampulla or the bursa.
- the embryos are implanted through this nick, and by holding onto the peritoneal wall, the reproductive organs are guided back into the abdominal cavity.
- the recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning.
- the weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.
- Genomic DNA is prepared from the tail snips using a Qiagen Dneasy kit following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to the human growth hormone (hGH) 3' UTR portion of the transgenic vector. A region unique to the human sequence was identified from an alignment of the human and mouse growth hormone 3' UTR DNA sequences, ensuring that the PCR reaction does not amplify the mouse sequence. Primers which amplify a 368 base pair fragment of hGH and primers which hybridize to vector sequences and amplify the cDNA insert, are often used along with the hGH primers. In these experiments, DNA from animals positive for the transgene will generate two bands, a 368 base pair band corresponding to the hGH 3' UTR fragment and a band of variable size corresponding to the cDNA insert.
- hGH human growth hormone
- mice may be back- crossed into an inbred strain by placing a TG female with a wild-type male, or a TG male with one or two wild-type female(s). As pups are born and weaned, the sexes are separated, and their tails snipped for genotyping.
- mice Three 4 week old male zalpha51 transgenics and an age-matched nontransgenic male control from the same litter were necropsied and their tissues microscopically evaluated. Prior to death, 2 of the mice were noticed to have abnormal locomotion. The two affected mice rapidly developed rigor mortis after being humanely euthanitized by anesthetic overdose. Following euthanasia, the mice were immediately necropsied and tissues collected into 10% neutral buffered formalin.
- tissue were routinely processed, sectioned at 5 ⁇ and stained with hematoxylina and eosin for histopathology: brain, spinal cord, skull including teeth, nasal passages, eye and Harderian gland, liver, heart, kidney, lung, thymus, spleen, mesenteric lymph node, salivary gland, pancreas, stomach, small and large intestine, accessory sex glands, prostate, vas deferens, epididymis, testis, pituitary, adrenal, trachea, esophagus, skin, skeletal muscle, sciatic nerve, femur and bone marrow. Tissues were evaluated under a light microscope (Nikon Eclipse E600, Nikon Corporation, Tokyo).
- the transgenics with ambulatory difficulties were found to have severe necrosis in the cerebellar folia (encephalomalacia).
- the necrotic cells were primarily located in the granular and Purkinje cell layers of the cerebellum.
- Acute perivasculitis was observed in the pons and cerebellar folia in both mice and in the ventral spinal cord of one of the mice. Both mice also had diffuse moderate lymphoid depletion in the thymus, necrosis of scattered acinar cells in the pancreas (common in transgenics with the metallothionein promoter) and minimal degeneration of the sciatic nerve.
- mice also had mild swelling of scattered fibers in the skeletal muscle of the rear leg (myopathy).
- myopathy No significant changes were found in the tissues of the nontransgenic control or third transgenic mouse.
- Expression analysis revealed that the two affected mice were high expressors while the third transgenic had no detectable expression of zalpha51.
- the microscopic changes found in the central nervous system were extremely uncommon in mice. The microscopic appearance of the lesions was similar to those observed in chicks with vitamin E deficiency. A form of spinocerebellar ataxia in humans has been associated with a severe deficiency of this vitamin. The rapid onset of rigor mortis in these transgenic mice suggested a metabolic derangement.
- Mitochondrial disorders can cause metabolism-related changes in a variety of tissue (e.g. the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke)).
- MELAS syndrome mitochondrial myopathy, encephalopathy, lactic acidosis and stroke
- zalpha51 may have some role in inducing apoptosis, based on the cell death observed in the cerebellum and other tissues.
- a panel of cDNAs from human tissues is screened for zalpha51 expression using PCR.
- the panel is made in-house and contained 94 marathon cDNA and cDNA samples from various normal and cancerous human tissues and cell lines is shown in Table 5, below.
- the cDNAs come from in-house libraries or marathon cDNAs from in-house RNA preps, Clontech RNA, or Invitrogen RNA.
- the marathon cDNAs are made using the marathon-ReadyTM kit (Clontech, Palo Alto, CA) and QC tested with clathrin primers, and then diluted based on the intensity of the clathrin band.
- RNA quality used for the libraries are tested for average insert size by PCR with vector oligonucleotides that are specific for the vector sequences for an individual cDNA library; (2) Standardization of the concentration of the cDNA in panel samples is achieved using standard PCR methods to amplify full length alpha tubulin or G3PDH cDNA using a 5' vector oligonucleotide and 3' alpha tubulin specific oligonucleotide primer or 3' G3PDH specific oligo primer; and (3) a sample is sequenced to check for possible ribosomal or mitochondrial DNA contamination.
- the panel is set up in a 96-well format that included a human genomic DNA (Clontech, Palo Alto, CA) positive control sample. Each well contains approximately 0.2-100 pg/ ⁇ l of cDNA.
- the PCR reactions are set up using appropriate oligonucleotides, TaKaRa Ex TaqTM (TAKARA Shuzo Co LTD, Biomedicals Group, Japan), and Rediload dye (Research Genetics, Inc., Huntsville, AL).
- the typical amplification is carried out as follows: 1 cycle at 94°C for 2 minutes, 35 cycles of 94°C for 30 seconds, 66.3°C for 30 seconds and 72°C for 30 seconds, followed by 1 cycle at 72°C for 5 minutes.
- PCR reaction product About 10 ⁇ l of the PCR reaction product is subjected to standard Agarose gel electrophoresis using a 4% agarose gel.
- the correct predicted DNA fragment size is observed in: (1) fetal liver; (2) normal tissues from liver, placenta, spinal cord, spleen, testis, and trachea; and (3) cancerous tissues from esophagus, stomach, kidney, liver, lung, ovary, and rectum.
- Northern data confirmed that expression of a 1.35 kb mRNA was highly expressed in liver.
- a panel of cDNAs from murine tissues was screened for mouse zalpha51 expression using PCR.
- the panel was made in-house and contained 72 marathon cDNA and cDNA samples from various normal and cancerous murine tissues and cell lines are shown in Table 6, below.
- the cDNAs came from in-house libraries or marathon cDNAs from in-house RNA preps, Clontech RNA (Clontech, Palo Alto, CA), or Invitrogen RNA (Invitrogen, Carlsbad, CA).
- the mouse marathon cDNAs were made using the marathon-Ready kit (Clontech) and quality control tested with mouse transferrin receptor primers, and then diluted based on the intensity of the transferrin band.
- RNA quality control To assure quality of the amplified library samples in the panel, three tests for quality control (QC) were run: (1) To assess the RNA quality used for the libraries, the in-house cDNAs were tested for average insert size by PCR with vector oligonucleotides that were specific for the vector sequences for an individual cDNA library; (2) Standardization of the concentration of the cDNA in panel samples was achieved using standard PCR methods to amplify full length alpha tubulin or G3PDH cDNA using a 5' vector oligonucleotides, and 3' alpha tubulin specific oligonucleotide primer, or 3' G3PDH specific oligo primer, and (3) a sample was sent to sequencing to check for possible ribosomal or mitochondrial DNA contamination.
- the panel was set up in a 96-well format that included a mouse genomic DNA (Clontech) positive control sample. Each well contained approximately 0.2-100 pg/ ⁇ l of cDNA.
- the PCR amplification used Advantage 2 Taq PolymeraseTM (Clontech), and Rediload dye (Research Genetics, Inc., Huntsville, AL). The amplification was carried out as follows: 1 cycle at 94°C for 2 minutes; 35 cycles of 94°C for 10 seconds,
- the DNA fragment for Adipocytes was excised and purified using a Gel Extraction Kit (Qiagen, Chatsworth, CA) according to manufacturer's instructions. Fragments were confirmed by sequencing to show that they were indeed mouse zalpha51.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
L'invention concerne des polypeptides à quatre hélices en faisceaux, des éléments et des procédés de fabrication de ces polypeptides, ainsi que des procédés d'utilisation de ces derniers. Ces polypeptides contiennent au moins neuf résidus d'acides aminés contigus des séquences SEQ ID NO: 2 et SEQ ID NO: 5, et peuvent être fabriqués en tant que fusion polypeptidique comprenant des séquences hétérologues, tels que des marques d'affinité. Les polypeptides et les polynucléotides codant ces polypeptides peuvent être utilisés dans une large gamme d'applications thérapeutiques, diagnostiques et de recherche.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US558459 | 1983-12-06 | ||
US52784300A | 2000-03-17 | 2000-03-17 | |
US527843 | 2000-03-17 | ||
US55845900A | 2000-04-25 | 2000-04-25 | |
PCT/US2001/008493 WO2001070986A2 (fr) | 2000-03-17 | 2001-03-16 | Proteine helicoidale zalpha51 |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1268804A2 true EP1268804A2 (fr) | 2003-01-02 |
Family
ID=27062529
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP01916704A Withdrawn EP1268804A2 (fr) | 2000-03-17 | 2001-03-16 | Proteine helicoidale zalpha51 |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1268804A2 (fr) |
AU (1) | AU2001243693A1 (fr) |
CA (1) | CA2402757A1 (fr) |
WO (1) | WO2001070986A2 (fr) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7148330B2 (en) | 1999-07-30 | 2006-12-12 | Schering Corporation | Binding compounds for IL-27 |
WO2002079248A2 (fr) * | 2000-11-17 | 2002-10-10 | Zymogenetics, Inc. | Proteine 53 alpha helicoidale secretee par des mammiferes |
WO2005044850A1 (fr) * | 2003-10-29 | 2005-05-19 | Zymogenetics, Inc. | Proteine helicoidale zcyto32 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU1908599A (en) * | 1997-12-10 | 1999-06-28 | Zymogenetics Inc. | Mammalian alpha helical protein-1 |
PT1200592E (pt) * | 1999-07-30 | 2009-12-02 | Schering Corp | Citoquinas de mamífero; reagentes relacionados |
-
2001
- 2001-03-16 AU AU2001243693A patent/AU2001243693A1/en not_active Abandoned
- 2001-03-16 EP EP01916704A patent/EP1268804A2/fr not_active Withdrawn
- 2001-03-16 WO PCT/US2001/008493 patent/WO2001070986A2/fr not_active Application Discontinuation
- 2001-03-16 CA CA002402757A patent/CA2402757A1/fr not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO0170986A3 * |
Also Published As
Publication number | Publication date |
---|---|
CA2402757A1 (fr) | 2001-09-27 |
AU2001243693A1 (en) | 2001-10-03 |
WO2001070986A3 (fr) | 2002-03-14 |
WO2001070986A2 (fr) | 2001-09-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7241870B2 (en) | Interferon-like protein Zcyto21 | |
EP1988102B1 (fr) | Facteur de croissance homologue ZVEGF3 | |
WO1999055869A1 (fr) | Nouveaux facteurs et materiels de croissance polypeptidiques et leurs procedes de production | |
US6406888B1 (en) | Helical cytokine zalpha33 | |
WO2001070986A2 (fr) | Proteine helicoidale zalpha51 | |
US6531576B1 (en) | Four-helical bundle protein zsig81 | |
WO2001012665A2 (fr) | Cytokine zalpha48 a enroulement en helice | |
US20020009775A1 (en) | Helical protein zalpha51 | |
EP1181369B1 (fr) | Proteine en faisceau a quatre helices zsig81 | |
US6756214B2 (en) | Protein zlmda33 | |
US20030153050A1 (en) | Helical polypeptide zalpha29 | |
US20030022316A1 (en) | CUB domain protein zcub3 and materials and methods for making it | |
CA2387371A1 (fr) | Cytokine helicoidale zalpha33 | |
US20020192750A1 (en) | Neuropilin homolog zcub5 | |
CA2377580A1 (fr) | Polypeptide spirale zalpha29 | |
WO2001018205A1 (fr) | Polypeptide secrete zalpha30 | |
WO1999042583A1 (fr) | Homologues du facteur de croissance pour le tissu conjonctif | |
WO2001029223A2 (fr) | Proteine de membrane ztsl1 de type 1 | |
WO2005044850A1 (fr) | Proteine helicoidale zcyto32 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20021018 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR |
|
AX | Request for extension of the european patent |
Free format text: AL;LT;LV;MK;RO;SI |
|
R17P | Request for examination filed (corrected) |
Effective date: 20021016 |
|
17Q | First examination report despatched |
Effective date: 20040120 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20040521 |